Recombinant vector expressing multiple costimulatory molecules and uses thereof

ABSTRACT

The present invention is a recombinant vector encoding and expressing at least three or more costimulatory molecules. The recombinant vector may additionally contain a gene encoding one or more target antigens or immunological epitope thereof. The synergistic effect of these costimulatory molecules on the enhanced activation of T cells is demonstrated. The degree of Tell activation using recombinant vectors containing genes encoding three costimulatory molecules was far greater than the sum of recombinant vector constructs containing one costimulatory molecule and greater than the use of two costimulatory molecules. Results employing the triple costimulatory vectors were most dramatic under conditions of either low levels of first signal or low stimulator to T-cell ratios. This phenomenon was observed with both isolated CD4 +  and CD8 +  T cells. The recombinant vectors of the present invention are useful as immunogenes and vaccines against cancer and pathogenic micro-organisms, and in providing host cells, including dendritic cells and splenocytes with enhanced antigen-presenting functions.

FIELD OF THE INVENTION

The present invention relates to a recombinant vector comprising foreigngenes encoding multiple costimulatory molecules and optionally a foreigngene encoding a target antigen. The invention further relates to arecombinant virus comprising foreign genes encoding at least threecostimulatory molecules and optionally a foreign gene encoding at leastone target antigen or immunological epitope thereof. More specifically,the present invention relates to a recombinant poxvirus comprisingforeign genes encoding at least the costimulatory molecules: onemolecule from the B7 family, LFA-3 and ICAM-1 and optionally a foreigngene encoding at least one target antigen or immunological epitopethereof and uses thereof as immunogens and vaccines. The inventionfurther relates to antigen presenting cells transfected, infected ortransduced by a recombinant vector comprising foreign genes encodingmultiple costimulatory molecules and optionally a foreign gene encodingat least one target antigen or immunological epitope thereof.

BACKGROUND OF THE INVENTION

The extent of the primary response of T cells, which involves theiractivation, expansion, and differentiation, is paramount to a successfulimmune response to an antigen. The initiation of an immune responserequires at least two signals for the activation of naive T cells byantigen presenting cells (APC) (1-5). The first signal is antigenspecific, delivered through the T-cell receptor via the peptide/majorhistocompatibility complex, and causes the T cell to enter the cellcycle. The second, or “costimulatory,” signal is required for cytokineproduction and proliferation. At least three distinct molecules normallyfound on the surface of professional APC have been proposed as capableof providing the second signal critical for T-cell activation: B7.1(CD80), Intercellular adhesion molecule-1 (ICAM-1; CD54), and Leukocytefunction-associated antigen-3 (LFA-3; human CD58; murine CD48) (2, 6,7). The T-cell ligands for these costimulatory molecules are distinct.B7-1 interacts with the CD28 and CTLA-4 molecules, ICAM-1 interacts withthe CD11a/CD18 (LFA-1/2 integrin) complex, and LFA-3 interacts with theCD2 (LFA-2) molecules. It is not known whether these costimulatorymolecules perform equivalent functions or carry out specializedfunctions at specific stages of an induced immune response (2). Thesemolecules have been individually shown to costimulate T-cellproliferation in vitro (6). However, because they may be expressedsimultaneously on APC, it has been difficult to examine relativepotencies of individual costimulatory molecules during the induction ofT-cell proliferation (2).

As it has been proposed that both antigen and costimulatory moleculesmust be expressed in proximity to each other to properly co-engage the Tcell and costimulatory receptors (8, 9), the admixture of severalrecombinant viruses could be utilized to explore the potentialcooperation of costimulatory molecules. The disadvantage of thisapproach, however, is that the admixture of three or more viruses has astatistically diminished probability of co-infecting the same cell,thereby making a multi-gene construct much more desirable for use withmultiple costimulatory molecule genes.

WO 91/02805, published Mar. 7, 1991, discloses a recombinant retrovirusvector construct which directs the expression of a target antigen, anMHC protein and other proteins involved in immune interactions which aremissing or under-represented in a target cell.

Akagi, et al. 1997, J. Immunotherapy Vol. 20 (1):38-47 disclose anadmixture of a recombinant vaccinia virus containing a modified MUC1gene (rV-MUC1), and a recombinant vaccinia virus containing the gene forthe murine costimulatory molecule B7 (rV-B7).

Cavallo, P. et al. 1995, Eur. J. Immunol., 25:1154-1162 disclose thattransfection of B7-1 cDNA into three ICAM-1⁺ tumor cell lines issufficient to induce rejection in syngeneic mice.

Chen, L. et al. 1994, J. Exp. Med., 179:523-532 disclose a recombinantretrovirus vector containing cDNA for murine B7 and the use of thevector in transducing various tumors.

Damle, N. K. et al 1992, J. Immunol. Vol 148 (No. 7): 1985-1992 disclosethe use of an antigen presenting cell (APC)-independent in vitro culturesystem consisting of immobilized combinations of monoclonal antibodiesdirected at the TCR/CD3 complex and soluble Ig chimeras (RG) of fourdistinct APC—associated costimulatory molecules to compare the abilitiesof these molecules to costimulate T cell proliferation.

Dubey, C. et al 1995, J Immunol 155: 45-57 disclose a study of therelative contribution of ICAM-1: LFA-1 and B7: CD28/CTLA-4 costimulatorypathways in naïve T cell activation, using either anti-CD28 antibody orfibroblast cell lines transfected with I-E^(k), which express either nocostimulatory molecules, ICAM-1 alone, B7-1 alone, or ICAM-1 and B7-1together.

Fenton, R. G. et al, 1998 Vol. 21, No. 2, pp 95-108, disclosetransfection of the costimulatory molecule B7-1 gene into threeHLA-A2-expressing human melanoma cell lines, and their capacity tostimulate primary human T cells. The three melanoma lines also expresseddetectable levels of the costimulatory molecules ICAM-1 (CD54) and LFA-3(CD58).

Gjorloff Wingren, A. et al 1995, Critical Reviews in Immunol 15 (3 & 4):235-253 disclose that with co-transfection of HLA-DR, B7 and LFA-3 intoCHO cells, these molecules cooperate in activation of both naïve andmemory T cells and allow responses at picomolar concentrations of theantigen, staphylococcal enterotoxin B (SEB).

Goldbach-Mansky, R. et al 1992, International Immunol. 4 (No. 12):1351-1360 disclose that CD4 T cells respond to staphylococcalenterotoxin B (SEB) in the presence of the LFA-3, ICAM-1 and B7 positiveerythroleukemic cell line K562, murine L cells, and human B7 transfectedL cells.

Hodge, J. W. et al 1994, Cancer Research 54:5552-5555 disclose theconstruction and characterization of recombinant vaccinia virusescontaining the murine B7.1 and B7.2 genes.

Hodge, J. W. et al 1995, Cancer Research 55: 3598-3603 Cancer Research55:3598-3603 disclose an admixture of recombinant vaccinia murine B7.1(rV-B7) plus recombinant vaccinia expressing the human carcinoembryonicantigen gene (rV-CEA) and the use of this admixture for anti-tumoractivity.

Parra, et al 1993, Scand J. Immunol. 38: 508-514, Parra, E. et al 1994,J. Immunol 153: 2479-2487, and Parra, et al. 1997, J. Immunol.,458:637-642 disclose CHO cells transfected with the human HLA-DR4molecule (CHO-DR4); HLA-DR4 and B7 (CHO-DR4/B7), HLA-DR4 and LFA-3(CHO-DR4/LFA3); HLA-DR4 and ICAM-1 (CHO-DR4/ICAM-1); or DR4, B7 andLFA-3 (CHO-DR4/B7/LFA-3) genes.

Thomas, R. et al. 1993 J. Immunol. 151:6840-6852 disclose that freshlyobtained dendritic cells (DC) express similar densities of HLA-DR andthe accessory molecules LFA-3, ICAM-1 and B7 as monocytes.

Uzendoski, K et al. May 1997, Human Gene Therapy 8:851-860 disclose theconstruction, characterization and immunological consequences of arecombinant vaccinia virus expressing the murine costimulatory molecule,ICAM-1.

WO 96/10419, published Apr. 11, 1996, of PCT/US95/12624 disclosessubject matter relating to a single recombinant viral vector which hasincorporated one or more genes or portion thereof encoding animmunostimulatory molecule and one or more genes or portion thereofencoding an antigen of a disease state.

Robinson et al U.S. Pat. No. 5,738,852 discloses a retroviral vectorcontaining a polynucleotide sequence encoding a target antigen of aninfectious agent and a polynucleotide sequence encoding a B7costimulatory molecule.

The present invention is a vector containing foreign DNA encoding atleast three costimulatory molecules, alone or in combination withforeign DNA encoding at least one target antigen or immunologicalepitope thereof which allows functional expression of each foreign DNAin an infected host cell.

SUMMARY OF THE INVENTION

The present invention provides a recombinant vector comprising foreignor exogenous genes or portions thereof encoding multiple costimulatorymolecules. Genes or functional portions thereof encoding costimulatorymolecules having utility in the present invention include but are notlimited to a B7 family member, ICAM-1, LFA-3, 4-1B3BL, CD59, CD40, CD70,VCAM-1, OX-40L, functional portions and homologs thereof. The vector ofthe invention may further provide a foreign gene encoding at least onetarget antigen or immunological epitope thereof in combination with theforeign genes encoding multiple costimulatory molecules. The foreigngene encoding at least one target antigen or immunological epitopethereof may be derived from cells, tissues or organisms such as viruses,bacteria, protozoans, parasites, yeast, tumor cells, preneoplasticcells, hyperplastic cells, tissue specific cells, or synthetic antigens.The vector may further provide a foreign gene encoding at least one or acombination of cytokines, chemokines and flt-3L.

The recombinant vector for use in the present invention group consistingof bacterial vectors, virus vectors, nucleic acid based vectors and thelike. The recombinant virus vectors include but are not limited topoxvirus, adenovirus, herpes virus, alphavirus, retrovirus,picornavirus, iridovirus and the like. The poxvirus include but are notlimited to the orthopox, avipox, suipox and capripox.

The present invention provides a recombinant virus comprising foreigngenes or portions thereof encoding multiple costimulatory molecules forproviding an enhanced immune response to a target cell, target antigenor immunological epitope thereof which is greater than a responseprovided by a recombinant virus comprising a foreign gene or genesencoding single or double costimulatory molecules. The recombinant virusof the invention may further provide a foreign gene encoding at leastone target antigen or immunological epitope thereof in combination withthe foreign genes encoding multiple costimulatory molecules. Therecombinant virus may further provide a foreign gene encoding otherclasses of immunostimulatory molecules such as cytokines including butnot limited to IL-2, IL-12, GM-CSF and the like, chemokines such asMIP1, MIP2, RANTES and the like, and Flt-3L which stimulates DCproliferation.

The present invention further provides a recombinant poxvirus comprisingforeign genes or portions thereof encoding multiple costimulatorymolecules for providing an enhanced immune response to a target cell,target antigen or immunological epitope thereof which is greater than aresponse provided by a recombinant poxvirus comprising a foreign gene orgenes encoding single or double costimulatory molecules. The recombinantpoxvirus of the invention may further provide a foreign gene encoding atleast one target antigen or immunological epitope thereof in combinationwith the foreign genes encoding multiple costimulatory molecules.

The present invention also provides a recombinant poxvirus comprising anucleic acid sequence encoding and expressing multiple costimulatorymolecules, said nucleic acid sequence comprising a nucleic acid sequenceencoding at least one molecule from the B7 family of costimulatorymolecules, a nucleic acid sequence encoding an ICAM-1 costimulatorymolecules, and a nucleic acid sequence encoding an LFA-3 costimulatorymolecule. The recombinant virus further provides a multiplicity ofpoxvirus promoters which regulate expression of each foreign gene.

The present invention provides a recombinant virus produced by allowinga plasmid vector comprising foreign DNA encoding multiple costimulatorymolecules to undergo recombination with a parental virus genome toproduce a recombinant virus having inserted into its genome the foreignDNA. The recombinant virus produced by recombination may further containa foreign gene encoding at least one target antigen or immunologicalepitope thereof provided by the plasmid vector.

The present invention also provides a recombinant poxvirus produced byallowing a plasmid vector comprising foreign DNA encoding thecostimulatory molecule, LFA-3, ICAM-1 and at least one molecule from theB7 family to undergo recombination with a parental poxvirus genome toproduce a recombinant poxvirus having inserted into its genome theforeign DNA and a multiplicity of poxvirus promoters capable ofcontrolling the expression of the foreign DNA. The recombinant poxvirusproduced by recombination may further contain a foreign gene encoding atleast one target antigen or immunological epitope thereof provided bythe plasmid vector.

An object of the invention is to provide an immunogen for enhancement ofimmune responses against target cells, target antigens or immunologicalepitopes thereof comprising a recombinant vector having foreign nucleicacid sequences encoding multiple costimulatory molecules. The vector mayfurther comprise a foreign nucleic acid sequence encoding at least onetarget antigen or immunological epitope thereof.

Another object of the invention is to provide an immunogen forenhancement of immune responses against target cells, target antigens orimmunological epitopes thereof comprising a recombinant virus vectorhaving foreign nucleic acid sequences encoding three or morecostimulatory molecules. The recombinant virus vector may furthercomprise a foreign nucleic acid sequence encoding at least one or moretarget antigens or immunological epitopes thereof.

Yet another object of the invention is to provide an immunogen forenhancement of immune responses against target cells, target antigens orimmunological epitopes thereof comprising a recombinant poxvirus vectorcomprising a foreign nucleic acid sequence encoding the costimulatorymolecules LFA-3, ICAM-1 and at least one molecule from the B7 family anda foreign nucleic acid sequence encoding at least one target antigen orimmunological epitope thereof.

The vector of the present invention provides a vaccine for eliciting andenhancing immune responses against target cells, target antigens orepitopes thereof for protection and/or treatment of disease states. Thevector vaccine comprises foreign nucleic acid sequences encodingmultiple costimulatory molecules. The vector vaccine may also compriseforeign nucleic acid sequences encoding one or more target antigens orimmunological epitopes thereof for producing a monovalent or polyvalentvaccine against a disease.

The present invention provides pharmaceutical compositions comprising avector having foreign nucleic acid sequences encoding multiplecostimulatory molecules and a pharmaceutically acceptable carrier. Thevector may further comprise a foreign nucleic acid sequence encoding atleast one target antigen or immunological epitope thereof. The vectormay additionally comprise a nucleic sequence encoding a cytokine,chemokine, flt-3L, or combination thereof.

The present invention provides a pharmaceutical composition comprising arecombinant virus vector which comprises foreign or exogenous genes orfunctional portions thereof encoding three or more costimulatorymolecules, a foreign gene encoding at least one target antigen orimmunological epitope thereof, and a pharmaceutically acceptablecarrier.

The present invention also provides pharmaceutical compositionscomprising a recombinant poxvirus comprising foreign genes or portionsthereof encoding multiple costimulatory molecules and a pharmaceuticallyacceptable carrier. The recombinant poxvirus may further comprise aforeign nucleic acid sequence encoding at least one target antigen orimmunological epitope thereof.

Another aspect of the invention is a pharmaceutical compositioncomprising a recombinant poxvirus comprising foreign genes or portionsthereof encoding three or more costimulatory molecules, and may furthercomprise a foreign gene or portion thereof encoding at least one targetantigen or immunological epitope thereof, and a pharmaceuticallyacceptable carrier or immunological epitope thereof.

The present invention also provides a pharmaceutical compositioncomprising a first vector comprising foreign genes or functionalportions thereof encoding multiple costimulatory molecules and a secondvector comprising foreign genes encoding at least one target antigen orimmunological epitope thereof and a pharmaceutically acceptable carrier.

The present invention provides host cells infected, transfected ortransduced with a first vector comprising foreign genes encodingmultiple costimulatory molecules causing expression of the multiplecostimulatory molecules in the host cells. The first vector or a secondvector may further provide a foreign gene encoding at least one targetantigen or immunological epitope thereof to the host cell.

The present invention provides antigen-presenting cells (APCs) or tumorcells infected, transfected or transduced with a first vector comprisingforeign or exogenously provided genes encoding multiple costimulatorymolecules causing expression or overexpression of the multiplecostimulatory molecules. The first vector or a second vector may furtherprovide a foreign gene encoding at least one target antigen orimmunological epitope thereof to the host cell.

The present invention further provides host cells infected with arecombinant poxvirus causing expression of the multiple costimulatorymolecules, and optionally causing expression of a target antigen orimmunological epitope thereof.

Another aspect of the invention is a dendritic cell (DC) and precursorthereof infected, transfected or genetically engineered to overexpressgenes encoding multiple exogenous costimulatory molecules. The DCs andprecursors thereof may further be engineered to express foreign genesencoding at least one target antigen or immunological epitope thereof.

Yet another aspect of the invention is a DC and precursors thereofgenetically engineered to overexpress genes encoding at least threeexogenous costimulatory molecules. The DCs and precursor thereof mayfurther be engineered to express foreign genes encoding at least onetarget antigen or immunological epitope thereof.

The present invention further provides a DC and precursors thereofgenetically engineered to overexpress genes encoding at least one B7molecule, ICAM-1 and LFA-3. The DCs and precursor thereof may further beengineered to express foreign genes encoding at least one target antigenor immunological epitope thereof.

The present invention provides methods and a plasmid vector forrecombination with a parental virus designed to produce a recombinantvirus capable of expressing foreign nucleic acid sequences encodingmultiple costimulatory molecules comprising (a) a multiplicity of viralpromoters, (b) the foreign nucleic acid sequences encoding the multiplecostimulatory molecules, (c) DNA sequences flanking the constructs ofelements (a) and (b), the flanking sequences at both the 5′ and 3′ endsbeing homologous to a region of a parental virus genome where elements(a) and (b) are to be inserted. The plasmid vector may further provide aforeign nucleic acid sequence encoding at least one target antigen orimmunological epitope thereof. The plasmid vector may also provide agene encoding a selectable marker.

The present invention also provides methods and a plasmid vector forrecombination with a parental poxvirus designed to produce a recombinantpoxvirus capable of expressing foreign nucleic acid sequences encodingthe costimulatory molecules LFA-3, ICAM-1 and at least one B7 moleculewhich comprises (a) a multiplicity of poxviral promoters, (b) theforeign nucleic acid sequences encoding the LFA-3, ICAM-1 and at leastone B7 molecule, (c) DNA sequences flanking the construct of elements(a) and (b), the flanking sequences at both 5′ and 3′ ends beinghomologous to a region of a parental poxvirus genome where elements (a)and (b) are to be inserted. The plasmid vector may further provide aforeign nucleic acid sequence encoding at least one target antigen orimmunological epitope thereof. The plasmid vector may also provide agene encoding a selectable marker.

One aspect of the invention is a method of enhancing immunologicalresponses in a mammal to at least one target cell, target antigen orimmunological epitope thereof comprising administration of a firstvector comprising foreign nucleic acid sequences encoding multiplecostimulatory molecules, each costimulatory molecule expressed in a cellin the mammal in an amount effective to enhance at least oneimmunological response in the mammal. Genes or functional portionsthereof encoding costimulatory molecules having utility in the presentinvention include but are not limited to a B7 family member, ICAM-1,LFA-3, 4-1BBL, CD59, CD40, CD70, VCAM-1, OX-40L and homologs andportions thereof. A foreign nucleic acid sequence encoding at least onetarget antigen or immunological epitope thereof may further be providedin the method by the first vector or by a second vector.

In addition to genes or portion thereof encoding multiple costimulatorymolecules, a foreign or exogenous nucleic acid sequence or functionalportions thereof encoding at least one or a combination of other classesof immunostimulatory molecules may also be provided by the first vector,by the second vector, or by a third vector. Other classes ofimmunostimulatory molecules includes cytokines such as IL-2, IL-12,GM-CSF and the like, chemokines such as MIP1, MIP2, RANTES and the likeand Flt-3L.

An aspect of the invention is a method of enhancing an antigen-specificT cell immune response in a mammal to a target cell, target antigen orimmunological epitope thereof comprising administration of a foreignrecombinant poxvirus comprising nucleic acid sequences encoding multiplecostimulatory molecules LFA-3, ICAM-1 and at least one B7 molecule, eachcostimulatory molecule expressed in a cell in the mammal in an amounteffective to enhance at least one T-cell immune response in which theenhancement is greater than the additive sum of enhancement provided byadministration of single or double costimulatory molecules.

In another method of enhancing immunological responses, APCs or tumorcells expressing foreign or exogenously provided genes encoding multiplecostimulatory molecules are provided to a mammal in an effective amountto enhance immunological responses. The APC or tumor cell may furtherexpress foreign genes encoding at least one target antigen orimmunological epitope thereof for enhancement of immune responses. Atarget antigen or immunological epitope thereof may be administered tothe mammal prior to, concurrently with or subsequent to theadministration of the APC or tumor cell. In addition, or alternatively,APCs or tumor cells are pulsed with at least one target antigen orimmunological epitope thereof prior to administration to the mammal.

The present invention provides methods of enhancing humoral responses ina mammal to a target cell, target antigen or immunological epitopethereof comprising administration of a recombinant vector comprisingforeign nucleic acid sequences encoding multiple costimulatory moleculesto a mammal in an amount effective to enhance an humoral response. Thevector may further comprise nucleic acid sequences encoding at least onetarget antigen or immunological epitope thereof. The invention furtherprovides an isolated antibody or functional portion thereof against atarget cell, target antigen or immunological epitope thereof produced bythe method.

The present invention also provides antibody specific for a targetantigen or immunological epitope thereof produced in response toadministration of a recombinant poxvirus comprising foreign genesencoding B7, ICAM-1 and LFA-3 and genes encoding one or more targetantigens or epitopes thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thedetailed description of the invention.

FIG. 1. Genomic structure of plasmid pT5032 comprising nucleic acidsequences encoding murine LFA-3, ICAM-1 and B7.1, flanked by portions ofthe Hind III M region of the vaccinia genome.

FIG. 2. Genomic structure of plasmid pT5047 comprising nucleic acidsequences encoding murine LFA-3, ICAM-1, B7.1, and the lacZ gene,flanked by portions of the Hind III J region of the vaccinia genome.

FIG. 3. Genomic structure of plasmid pT5031 comprising nucleic acidsequences encoding murine LFA-3, ICAM-1 and B7.1 and a nucleic acidsequence encoding CEA, flanked by portions of the Hind III M region ofthe vaccinia genome.

FIGS. 4A through 4C. Genomic structure of recombinant vaccinia virusesexpressing three murine costimulatory molecules with (FIG. 4C) orwithout (FIGS. 4A and B) a tumor-associated antigen. FIG. 4A shows thegenomic structure of recombinant vaccinia, vT171. FIG. 4B shows thegenomic structure of recombinant vaccinia vT199. FIG. 4C shows thegenomic structure of recombinant vaccinia vT172. Hind III M and Hind IIIJ are the sites of insertion in the poxvirus genomes of the foreigngenes. Promoters 30K, 13, sE/L, 7.5K, 40K and C1 are poxyiral promoters.Bam HI and Hind III restriction sites in the inserted sequences areshown, with the distance of each site (in kilobase pairs) from the 5′end of the insertion (0) listed above each site in parentheses (notdrawn to scale).

FIG. 5. Genomic structure of plasmid pT8001 comprising nucleic acidsequences encoding murine B7.1, LFA-3, ICAM-1 and the lacZ gene, flankedby portions of the BamHI J region of the fowlpox genome.

FIG. 6. Genomic structure of plasmid pT5049 comprising a nucleic acidsequence encoding the tumor associated antigen, CEA, and murine B7.1,LFA-3, and ICAM-1, in combination with the lacZ gene, flanked byportions of the BamHI J region of the fowlpox genome.

FIGS. 7A through 7D. Genomic structure of recombinant fowlpox virusesexpressing three murine costimulatory molecules with (FIGS. 7B, 7C and7D) or without (FIG. 7A) a tumor-associated antigen (TAA). FIG. 7A showsthe genomic structure of recombinant fowlpox vT222. FIG. 7B shows thegenomic structure of recombinant fowlpox vT194. FIG. 7C shows thegenomic structure of recombinant fowlpox expressing MUC-1, B7.1, ICAM-1and LFA-3. FIG. 7D shows the genomic structure of recombinant fowlpoxexpressing a tumor-associated antigen, B7.1, ICAM-1 and LFA-3. BamHI Jis the site of insertion in the fowlpox virus genome of the foreigngenes. sE/L, 13, 7.5K, C1, 40K and 30 K are poxyiral promoters. P1-P5denote five different poxvirus promoters. BamHI and HindIII restrictionsites in the inserted sequences are shown, with the distance of eachsite (in kilobase pairs) from the 5′ end of the insertion (0) listedabove each site in parentheses (not drawn to scale).

FIG. 8. Genomic structure of plasmid pT5064 comprising nucleic acidsequences encoding human LFA-3, human ICAM-1, human B7.1 and the lacZgene, flanked by portions of the HindIII J region of the vacciniagenome.

FIGS. 9A through 9C Genomic structure of recombinant poxvirus expressingthree human costimulatory molecules LFA-3, ICAM-1 and B7.1 along withthe lacZ gene with (FIG. 9B, C) or without (FIG. 9A) a tumor associatedantigen, HindIII J is the site of insertion in the vaccinia virus genomeof the foreign genes. BamHI J is the site of insertion in the fowlpoxvirus genome. 30K, I3, sE/L, 40K and C1 are poxyiral promoters. BglIIand HindIII restriction sites in the inserted sequences are shown, withthe distance of each site (in kilobase pairs) from the 5′ end of theinsertion (0) listed above each site in parentheses (not drawn toscale).

FIG. 10. Genomic structure of plasmid pT8016 comprising nucleic acidsequences encoding CEA (6D) and human LFA-3, ICAM-1, B7.1, and the E.coli lacZ gene, flanked by portions of the HindIII J region of thevaccinia genome.

FIG. 11. Genomic structure of recombinant vaccinia virus vT238expressing CEA (6D) and three human costimulatory molecules. HindIII Jis the site of insertion in the poxvirus genome of the foreign genes.40K, 30K, 13, sE/L, and C1 are poxyiral promoters.

FIG. 12. Genomic structure of plasmid pT8019 comprising nucleic acidsequences encoding murine LFA-3, ICAM-1, B7.1, and the E. coli lacZgene, flanked by portions of the BamHI J region of the fowlpox genome.

FIGS. 13A and 13B. Genomic structure of recombinant fowlpox virusesexpressing murine or human costimulatory molecules. FIG. 13A shows thegenomic structure of recombinant fowlpox vT251. FIG. 133B shows thegenomic structure of recombinant fowlpox vT232. BamHI J is the site ofinsertion in the poxvirus genome of the foreign genes. 30K, 13, sE/L andC1 are poxyiral promoters.

FIG. 14. Genomic structure of plasmid pT5072 comprising nucleic acidsequences encoding human LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene,flanked by portions of the BamHI J region of the fowlpox genome.

FIG. 15. Genomic structure of plasmid pT8020 comprising nucleic acidsequences encoding MUC-1, murine LFA-3, ICAM-1, B7.1, and the E. colilacZ gene, flanked by portions of the BamHI J region of the fowlpoxgenome.

FIG. 16A through 16D. Genomic structure of recombinant fowlpox virusesexpressing murine or human costimulatory molecules with at least onetumor-associated antigen. FIG. 16A shows the genomic structure ofrecombinant fowlpox vT250. FIG. 166B shows the genomic structure ofrecombinant fowlpox vT242. FIG. 16C shows the genomic structure ofrecombinant fowlpox vT236. FIG. 16D shows the genomic structure ofrecombinant fowlpox vT257. BamHI J is the site of insertion in thepoxvirus genome of the foreign genes. 40K, 7.5K, 30K, 13, sE/L, and C1are poxyiral promoters.

FIG. 17. Genomic structure of plasmid pT2186 comprising nucleic acidsequences encoding MUC-1, human LFA-3, ICAM-1, B7.1, and the E. colilacZ gene, flanked by portions of the BamHI J region of the fowlpoxgenome.

FIG. 18. Genomic structure of plasmid pT2187 comprising nucleic acidsequences encoding CEA (6D), human LFA-3, ICAM-1, B7.1, and the E. colilacZ gene, flanked by portions of the BamHI J region of the fowlpoxgenome.

FIG. 19. Genomic structure of plasmid pT5080 comprising nucleic acidsequences encoding PSA, PSMA, human LFA-3, ICAM-1, B7.1, and the E. colilacZ gene, flanked by portions of the BamHI J region of the fowlpoxgenome.

FIG. 20. Genomic structure of plasmid pT5085 comprising nucleic acidsequences encoding murine LFA-3, ICAM-1, B7.1, and the E. coli lacZgene, flanked by portions of the deletion III region of the MVA genome.

FIGS. 21A and 21B. Genomic structure of recombinant MVA virusesexpressing murine or human costimulatory molecules with or withouttumor-associated antigens. FIG. 21A shows the genomic structure ofrecombinant MVA vT264. FIG. 21B shows the genomic structure ofrecombinant MVA vT260. Deletion III is the site of insertion in thepoxvirus genome of the foreign genes. 40K, 7.5K, 30K, 13, sE/L, and C1are poxyiral promoters.

FIG. 22. Genomic structure of plasmid pT5084 comprising nucleic acidsequences encoding PSA, PSMA, human LFA-3, ICAM-1, B7.1, and the E. colilacZ gene, flanked by portions of the deletion III region of the MVAgenome.

FIG. 23. Costimulatory molecule surface expression following infectionwith recombinant viruses. MC38 tumor cells were infected for 5 hours at5 MOI (multiplicity of infection; pfu/cell) with the indicated virus.After infection, cells were immunostained with FITC-labeled monoclonalantibodies (MAb) specific for the costimulatory molecule. Shaded areasare fluorescence intensity of the specific MAb while unshaded areas arethe fluorescence intensity of the appropriate isotype control antibody(see Materials and Methods).

FIGS. 24A and 24B. Effect of multiple costimulatory molecules on T-cellproliferation. Naive murine T cells, in the presence of varyingconcentrations of Con A to provide the first signal, were co-culturedwith MC38 stimulator cells infected with either recombinant vaccinia(FIG. 24A) or recombinant fowlpox (FIG. 24B) vectors. Recombinantvectors were wild-type (i.e., V-Wyeth or WT-FP [open squares]), rV-LFA-3(closed triangle), rV-ICAM-1 or rF-ICAM-1 (closed circles), rV-B7-1 orrF-B7-1 (closed diamonds), and rV-B7-1/ICAM-1/LFA-3 orrF-CEA/B7-1/ICAM-1/LFA-3 (closed squares). Uninfected MC38 cells areopen circles. Proliferation assay is as described in Materials andMethods.

FIGS. 25A through 25D. Specificity of costimulation delivered viarecombinant vaccinia viruses. T cells, in the presence of Con A, wereco-cultured with MC38 stimulator cells infected with V-Wyeth (FIG. 25A),rV-B7-1 (FIG. 25B), rV-ICAM-1 (FIG. 25C), and rV-LFA-3 (FIG. 25D), asdenoted by open circles. Infected stimulator cells in the presence ofcostimulatory molecule-specific MAb are denoted by closed circles, andisotype control antibody is denoted by closed triangles.

FIG. 26. Relative capacity of B7-1, ICAM-1, LFA-3 and the coexpressionof all three costimulatory molecules to deliver the second signal forT-cell proliferation. In the presence of Con A (2.5 μg/ml), 100,000 Tcells were co-cultured with 10,000 MC38 cells. The stimulator MC38 cellsexpressing one or all of the costimulatory molecules were added to thewells in various ratios in combination with V-Wyeth-infected stimulatorcells to a total of 10⁴ MC38 cells/well. MC38 cells were infected withV-Wyeth (open square), rV-LFA-3 (closed triangles), rV-ICAM-1 (closedcircles), rV-B7-1 (closed diamonds), or rV-B7-1-ICAM-1-LFA-3 (closedsquares). Cells were co-cultured for 48 hours. During the final 18hours, ³H-Thymidine was added to measure T-cell proliferation. Insetpanel depicts proliferation values obtained from a culture in which 3%of the MC38 stimulator cells were infected with the vectors shown. Thus,in this experiment, the final ratio of stimulator cells to T cells was0.003. Note the relatively poor effect of rVB7.1/ICAM under theseconditions as compared to rV-B7/ICAM/LFA-3.

FIGS. 27A through 27D. Effect of costimulation on specific T-cellpopulations. Murine CD4⁺ (FIG. 27A) or CD8⁺ T cells (FIG. 27B) wereco-cultured with uninfected MC38 cells (open circle), or cells infectedwith V-Wyeth (open squares), rV-LFA-3 (closed triangles), rV-ICAM-1(closed circles), rV-B7-1 (closed diamonds) or rV-B7-1/ICAM-1/LFA-3(closed squares) at a 10:1 ratio for 48 hours in the presence of variousconcentrations of Con A. During the final 18 hours, ³H-Thymidine wasadded to measure T-cell proliferation. FIGS. 27C and 27D show theproliferative responses of purified CD4⁺ and CD8⁺ cells, respectively,when co-cultured in the presence of vector-infected MC38 stimulatorcells at a low Con A concentration (0.625 μg/ml).

FIGS. 28A through 28D. Effect of costimulation on cytokine production.Murine CD4⁺ (FIGS. 28A and 28C) or CD8⁺ (FIGS. 28B and 28D) T cells werepurified as described in Materials and Methods and co-cultured with theindicated MC38 vector-infected stimulator cells for 24 hours in thepresence of 2.5 μg/ml Con A. Supernatant fluids were analyzed forproduction of IL-2 (FIGS. 28A and 28B) and IFN-γ (FIGS. 28C and 28D) bycapture ELISA.

FIGS. 29A through 29C. Effect of costimulation on cytokine RNAexpression. FIG. 29A: murine CD4⁺ or CD8⁺ T cells were co-cultured withMC38 stimulator cells infected with V-Wyeth (lane A), rV-B7-1 (lane B),rV-ICAM-1 (lane C), rV-LFA-3 (lane D) or rV-B7-1/ICAM-1/LFA-3 (lane E)at a T-cell to stimulator cell ratio of 10:1 for 24 hours in thepresence of 2.5 μg/ml Con A. Following culture, T-cell RNA was analyzedby multiprobe RNAse protection assay. The quantitative representation ofresults from the autoradiograph is normalized for expression of thehousekeeping gene L32 in FIG. 29B (CD4⁺ cells) and FIG. 29C (CD8⁺cells). Order of histogram bars (from left to right) is MC38/V-Wyeth,MC38/B7-1, MC38/ICAM-1, MC38/LFA-3, and MC38/B7-1/ICAM-1/LFA-3.

FIG. 30. C57BL/6 mice (5/group) were administered HBSS (closed squares)or vaccinated with 10⁷ pfu rV-CEA (closed triangles) or rV-CEA/TRICOM(closed circle). One hundred days later, mice were inoculated with1×10⁶MC38 carcinoma cells expressing CEA and survival was monitored. Allmice other than the rV-CEA/TRICOM group developed tumors and weresacrificed when tumors exceeded 20 mm in length or width, or when themice were moribund. FIG. 30: In a second experiment, C57BL/6 mice(5/group) were vaccinated with 10′ pfu rV-CEA, rV-CEA/B7.1,rV-CEA/TRICOM or HBSS buffer. Lymphoproliferative responses from pooledsplenic T cells were analyzed 22 days following vaccination. Valuesrepresent the stimulation index of the mean cpm of triplicate sames vs.media. Standard deviation never exceeded 10%. Antigens used were Con A(5 μg/ml), CEA (100 μg/ml) and ovalbumin (100 μg/ml).

FIG. 31 shows a schematic of an in vitro costimulation assay ofdendritic cells.

FIGS. 32A and 32B show the proliferative response of naïve CD4⁺ (FIG.32A) or naive CD8⁺ (FIG. 32B) T cells stimulated with progenitor DCsinfected with rV-B7/ICAM-1/LFA-3 or DCs (noninfected, i.e., CD 34⁺ cellstreated with GM-CSF+IL-4 for 6 days) in the presence of Con A.

FIGS. 33A and 33B show the proliferative response of naïve CD4⁺ (FIG.33A) or naïve CD8′ (FIG. 33B) T cells stimulated with progenitor DCsinfected with rV-B7/ICAM-1/LFA-3 or DCs infected with rV-B7/ICAM-1/LFA-3or V-Wyeth (control).

FIG. 34 shows the mixed lymphocyte reaction (MLR) of Balb/C splenocytesvs. irradiated C57bl/6 dendritic cells infected with 25 MOI of V-Wyethor rV-TRICOM. ³H-thymidine pulsed on day 3, harvest on day 4, □ DC(uninfected), ▪ DC (V-Wyeth infected), □ DC (rV-TRICOM infected).

FIG. 35 shows the proliferative response of responder T cells (CAP-M8T-cell line specific for CEA peptide 8) at various APC ratios harvestedon day 5 after stimulation with peptide-pulsed DCs infected withrV-TRICOM and rested 2 days with 10 u/ml IL2 (no APC or peptide).Peptide 8—(EAQNTTYL) in assay at 1 ug/ml final concentration.³H-thymidine added on day 2, T cells harvested on day 3.0=DC(v-Wyeth)−pep and Δ=DC (rV-TRICOM)—pep results are at baseline.

FIGS. 36A and 36B. Efficiency of poxyiral infection of murine dendriticcells (DC). DC were infected with 25 MOI rV-TRICOM or 50 MOI rFCEA/TRICOM for 5 h. DC infected with TRICOM vectors exhibit enhancedcapacity to stimulate naïve T-cells. All DC populations were co-culturedfor 48 h with T-cells at a ratio of 10:1 in the presence of differentconcentrations of Con A to provide signal-1. ³H-thymidine was addedduring the final 18 μl. FIG. 36A: Uninfected DC (closed squares),mock-infected DC (closed diamonds), or DC infected with V-WT (closedinverse triangles), rV-B7.1 (open triangles) or rV-TRICOM (opencircles). FIG. 36B: DC (closed squares), mock-infected DC (closeddiamonds), or DC infected with WT-FP (closed inverse triangles), rF-B7.1(open triangles) or rF-TRICOM (open circles).

FIGS. 37A through 37F. Enhanced allostimulatory activity by DC infectedwith vaccinia (FIGS. 37 A, C, E) or fowlpox (FIGS. 37 B, D, F) vectors.Uninfected DC (closed squares); mock-infected DC (closed diamonds); orDC infected with wild-type poxyiral vectors (V-WT or F-WT, closedinverse triangles), rV-B7.2 or rF-B7.1 (open triangles), or rV-TRICOM orrF-TRICOM (open circles) were co-cultured with allogeneic (FIGS. 37A-D)or syngeneic T cells (FIGS. 37E-F) for 5 days. ³H-thymidine was addedduring the final 18 h.

FIGS. 38A through 38F. Effect of vaccinia infection of DC onpeptide-specific T-cell proliferation. Uninfected DC (closed squares),or DC infected with V-WT (closed inverse triangles), rV-B7.1 (opentriangles) or rV-TRICOM (open circles) were co-cultured with OVApeptide-specific T cells (FIGS. 38A, C, E) or CAP-M8 peptide-specific Tcells (FIGS. 38 B, D, F). Experimental conditions included a fixedeffector:stimulator cell ration of 10:1 in the presence of variousconcentrations of the appropriate peptides (FIG. 38A-D), negativecontrol peptides (open squares, either VSVN (FIG. 38A), or FLU-NP (FIG.38B), or a fixed peptide concentration of 1 μM in the presence ofvarious effector:stimulator cell ratios (FIGS. 38E and F).

FIGS. 39A and 39B. Effect of rV-TRICOM infection with DC matured withTNF-α or CD40. DC (closed squares), or DC cultured with either 100 ng/mlTNF-α (open triangles), or 5 μg/ml CD40 mAb (open circles) for the final24 h of culture were used to stimulate CAP-M8-specific effector T cells(FIG. 39A). The proliferation of CAP-M8 T cells in response to these DCpopulations after infection with 25 MOI rV-TRICOM (FIG. 39B). For allpanels, the T-cell:DC ratio was 10:1, while the CAP-M8 peptideconcentration was 1 μg/ml. Closed circles denote proliferation of CAP-M8T cells stimulated with all DC populations in the presence of 1 μg/mlVSVN peptide.

FIGS. 40A through 40H: Effect of vaccinia infection of DC on inductionof CTL activity. DC (FIG. 40B), or DC infected with V-WT (FIG. 40C), orrV-TRICOM (FIG. 40D) were pulsed with 10 μM OVA peptide for 2 h. DCpopulations were administered intravenously to mice (1×10⁵ cells/mouse).Control mice were immunized subcutaneously with 100 μg OVA peptide inRibi/Detox adjuvant (FIG. 40A). Fourteen days later spleens wereharvested, restimulated for 6 days with the corresponding peptide, andassessed for lytic ability against EL-4 cells pulsed with either OVA(closed squares) or VSVN peptides (open squares). Inset numbers depictCTL activity as expressed in lytic units. Also shown is the effect ofvaccinia infection of DC on induction of CTL activity. DC (FIG. 40F), orDC infected with V-WT (FIG. 40G), or rV-TRICOM (FIG. 40H) were pulsedwith 10 μM CAP-M8 peptide for 2 h. DC populations were administeredintravenously to mice (1×10⁵ cells/mouse). Control mice were immunizedsubcutaneously with 100 μg CAP-M8 peptide in Ribi/Detox adjuvant (FIG.40E). Fourteen days later spleens were harvested, restimulated for 6days with the corresponding peptide, and assessed for lytic abilityagainst EL-4 cells pulsed with either CAP-M8 (closed squares) or FLU-NPpeptides (open squares). Inset numbers depict CTL activity as expressedin lytic units.

FIGS. 41A through 41C: Effect of multiple immunizations withvaccinia-infected DC on induction of CTL activity. DC (closed squares),or DC infected with V-WT (closed inverse triangles) or rV-TRICOM (opencircles) were pulsed with 10 μM CAP-M8 peptide for 2 h. DC populationswere administered intravenously to mice (1×10⁵ cells/mouse) 1, 2 or 3times at 7 day intervals. Control mice were immunized subcutaneouslywith 100 μg CAP-M8 peptide in Ribi/Detxo adjuvant (crosses). Fourteendays after the final immunization, spleens were harvested, restimulatedfor 6 days with CAP-M8, and assessed for lytic ability against EL-4cells pulsed with CAP-M8 or control peptide VSVN (not shown).

FIGS. 42A and 42B. Effect of vaccinia and fowlpox TRICOM-infectedsplenocytes on T cell proliferation. Naïve murine T cells wereco-cultured with autologous splenocytes infected with either recombinantvaccinia or fowlpox vectors. Co-culture was performed in varyingconcentrations of Con-A as Signal-1. Recombinant vectors were wild type(i.e. V-WT, FP-WT, open diamond), rV-B7-1 or rF-B7-1, (open circles) orrV-TRICOM or rF-TRICOM (closed squares). Uninfected splenocytes areshown as open triangles.

FIGS. 43A through 43D. Effect of TRICOM vector infected splenocytes onallogeneic T cells. Naïve Balb/C T cells were co-cultured with C57B1/6splenocytes infected with recombinant vaccinia (FIGS. 43A and C) orfowlpox (FIGS. 43B and D) vectors for either 2 days (FIGS. 43A and B) or5 days (FIGS. 43C and 43D). Recombinant vectors were V-WT or FP-WT, opendiamonds, rV-B7-1 or rF-B7-1 (open circles), or rV-TRICOM or rF-TRICOM(closed squares). Uninfected splenocytes are indicated as opentriangles. Proliferation induced by DC is indicated as closed squares.

FIGS. 44A through 44F. Effect of rV-TRICOM-infected splenocytes onspecific T cell populations. Naïve murine T cells were fractionated withCD3⁺, CD4⁺, and CD8⁺ subpopulations. T cells were co-cultured witheither uninfected autologous BMDC or splenocytes infected withrecombinant vaccinia vectors. Varying Con-A concentrations (FIGS. 44A-C)or varying number of stimulator cells (FIG. 44D-F) provided the firstsignal. T cell proliferation in response to mature BMDC is indicated byopen squares, and to uninfected splenocytes by open triangles.Recombinant vectors were wild-type (V-WT, open diamonds) or rV-TRICOM(closed squares).

FIGS. 45A through 45F. Effect of rV-TRICOM-infected bone marrow cells onspecific T cell populations. Naïve murine T cells were fractionated intoCD3⁺, CD4⁺, and CD8⁺ subpopulations. T cells were co-cultured witheither uninfected autologous BMDC or splenocytes infected withrecombinant vaccinia vectors. Varying Con-A concentrations (FIG. 45A-C)or varying number of stimulator cells (FIG. 45D-F) provided the firstsignal. T cell proliferation in response to mature BMDC is indicated byopen squares, and to uninfected splenocytes by open triangles.Recombinant vectors were wild-type (V-WT, open diamonds) or rV-TRICOM(closed squares).

FIGS. 46A through 46D. Effect or rV-TRICOM-infected splenocytes or bonemarrow (BM) cells on peptide-specific memory CD8⁺ T cells.CAP-M8-specific T cells were co-cultured with autologous splenocytes(FIGS. 46A and B) or bone marrow cells (FIGS. 46C and D) infected withrecombinant vaccinia vectors. The analysis was carried out using twosets of conditions: a) a 10:1 fixed ratio of responder:stimulator cellsthat were cultured in the presence of several concentrations of CAP-M8peptide (FIGS. 46A and 46C), or b) a fixed concentration of peptide (1uM) at various responder:stimulator ratios (FIGS. 46B and 46D).Recombinant vectors were wild type (open diamonds), and rV-TRICOM(closed squares). Uninfected splenocytes are shown as open triangles. BMare shown as open squares.

FIG. 47. Shows production of IFN-γ by human T cells isolated fromperipheral blood mononuclear cells (PBMC) using rF-TRICOM-infected humandendritic cells pulsed with CEA peptides, CAP-1 or CAP1, 6D.

FIG. 48. Shows production of IFN-γ by human T cells usingrF-TRICOM-infected human dendritic cells pulsed with PSA peptide, PSA-3.

FIG. 49. Shows production of IFN-γ by human T cells isolated from PBMCusing rF-TRICOM-infected human dendritic cells pulsed with Flu peptide58-66.

FIG. 50. Shows production of IFN-γ by human T cells isolated from PBMCusing rF-TRICOM- or rF-B7.1-infected human dendritic cells pulsed withFlu peptide 58-66 at various effector:APC ratios.

FIG. 51. Shows production of IFN-γ by human T cells from donor 868 usingrF-TRICOM-infected human dendritic cells pulsed with HPV peptide (11-20)after one or two in vitro stimulation (IVS).

FIG. 52. Shows production of IFN-γ by human T cell line using rF-TRICOM-or rF-B7.1-infected human dendritic cells pulsed with HPV peptide(11-20).

FIG. 53. Shows production of IFN-γ by a human T cell line usingrF-TRICOM- or rF-B7.1-infected human dendritic cells pulsed with variousconcentrations of HPV peptide (11-20).

FIG. 54. Shows production of IFN-γ by human T cells using rF-TRICOM orrF-B7.1-infected human dendritic cells pulsed with HPV E7 peptide 11-20at various effector:APC ratios.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a recombinant vector comprising foreign genesencoding multiple costimulatory molecules, in combination, or thefunctionally active portions of each costimulatory molecule. Multiplecostimulatory molecules as used herein are at least three or morecostimulatory molecules. As used herein a functionally active portion isthat portion of the molecule responsible for binding to its respectiveligand, triggering an appropriate costimulatory signal for immune-cellactivation. One method of determining functional activity is to accessthe induction of naïve T-cell proliferation by delivering thecostimulatory molecule to a target cell in vitro as described herein. Afunctional portion of a costimulatory molecule stimulates at least 20%increase in T cell proliferation.

The term foreign gene or foreign nucleic acid sequence or functionalportion thereof as used herein is a gene, nucleic acid sequence orfunctional portion thereof that is exogenously provided by a recombinantvector to a host cell or organism. The exogenous gene or portion thereofwhich is provided to the host cell or host organism may be one which isnot endogenously present in the host cell or organism or may beendogenously present and functional or non-functional. In the case inwhich a functional endogenous gene is present in the host cell ororganism, the foreign or exogenously provided gene or functional portionthereof results in overexpression of the gene product.

The recombinant vectors of the present invention have utility inproviding enhanced immunological response to cells of the immune systemincluding but not limited to T lymphocytes, B lymphocytes, NK cells,antigen-presenting cells (APCs) and the like. The enhancement of theimmunological response using the recombinant vectors expressing multiplecostimulatory molecules is synergistic as compared to the use of asingle costimulatory molecule or the use of two costimulatory moleculesin enhancing immunological responses. The immunological response may bea cellular and/or humoral immune response and may be directed to aspecific target antigen or epitope therefor may be a generalized immuneenhancing or upregulating effect as demonstrated by increased cytokinerelease, increase proliferation by immune cells, increased mitogenresponsiveness and the like. The enhancement in an immune responsepreferably includes hyperstimulation or high intensity T cellstimulation (HITS) as a result of stimulation using the recombinantvectors of the present invention or cells transfected, transduced orinduced by the recombinant vector of the present invention.

The foreign genes encoding the costimulatory molecules may be obtainedfrom a variety of sources. The selection of the source of foreign genesencoding the costimulatory molecules may depend on the species to beimmunized or treated using the recombinant vector.

The foreign genes encoding the costimulatory molecules may bemurine-derived, human-derived, simian-derived, other mammalian homologsand may be chemically synthesized based on mammalian genes. The foreigngenes encoding the costimulatory molecules may also be avian-derived orchemically synthesized based on avian costimulatory molecule genes. Therecombinant vectors of the present invention are useful as immunogensand as vaccines in stimulating an enhancement of immunological responsesto target cells, target antigens and immunological epitopes thereof.Such level of enhancement of a immune response using the presentrecombinant vectors comprising genes encoding multiple costimulatorymolecules has not been obtainable using a single or double costimulatorymolecule.

Genes or functional portions thereof encoding costimulatory moleculeshaving utility in the present invention include but are not limited toB7.1, B7.2, ICAM-1, LFA-3, 4-1 BBL, CD59, CD40, CD70, VCAM-1, OX-40L,mammalian homologs and the like. The recombinant vector of the presentinvention comprises genes encoding at least three costimulatorymolecules for synergistic enhancement of immune responses which is notobtainable by the use of a single or a double costimulatory molecule.Genes encoding various combinations of costimulatory molecules are anambit of the invention for use in the recombinant vector and may includesuch combinations as B7.1, B7.2, ICAM-1, LFA-3; B7.1, B7.2, ICAM-1,LFA-3; B7.1, B7.2, ICAM-1, 4-1BBL; B7.1, B7.2, ICAM-1, LFA-3, 4-1BBL;CD59, VCAM-1; and B7.1, B7.2; CD59, CD40, 4-1 BBL, CD70 and VCAM-1,B7.1, B7.2; OX-40L, 4-1BBL; and the like depending on the desired immuneresponse and the disease or condition to be treated. Based on thedramatic synergistic immune responses achieved using a recombinantvector encoding three costimulatory molecules as compared to the use ofa recombinant vector encoding one or two costimulatory molecules, arecombinant vector encoding four, five or more costimulatory moleculeswill result in a synergistic immune response or immune response equalto/or greater than that using a recombinant vector encoding threecostimulatory molecules.

B7 represents a family of costimulatory molecules which are members ofthe Ig gene superfamily. The members include murine B7.1 (CD80) and B7.2(CD86). B7.1 and B7.2 are the natural ligands of CD28/CTLA-4 (CD152).The gene sequence of murine B7.1 is disclosed in Freeman et al (J.Immunol. 143:2714-2722, 1989) and in GENBANK under Accession No. X60958.The gene sequence of murine B7.2 is disclosed in Azuma et al (Nature366:76-79, 1993) and in GENBANK under Accession No. L25606 and MUSB72X.

The human homologs of the murine B7 costimulatory molecules andfunctional portions thereof are an ambit of the present invention andhave particular utility in recombinant vectors for human clinical use.The human homolog of the murine B7 costimulatory molecules include CD80,the homolog of murine B7.1, and CD86, the homolog of B7.2. The genesequence of human B7.1 (CD80) is disclosed in GENBANK under AccessionNo. M27533, and the gene sequence of human B7.2 (CD86) is disclosedunder Accession No. U04343 and AF099105. A license may be required topractice this invention.

For use in the present invention, a recombinant vector may contain aforeign nucleic acid sequence encoding at least one molecule from the B7costimulatory molecule family, or a combination of B7 costimulatorymolecules or functional portions thereof in addition to othercostimulatory molecules. The combination of B7 costimulatory moleculesincludes but is not limited to two or more B7.1 molecules, two or moreB7.2 molecules, B7.1 and B7.2 and the like. In one embodiment therecombinant vector contains a foreign nucleic acid sequence encoding theB7.1 molecule in combination with foreign nucleic acid sequencesencoding LFA-3 and ICAM-1.

Intercellular adhesion molecule-1 (murine ICAM-1, CD54) and the humanhomolog, CD54, also acts as a costimulatory molecule. Its ligand isleukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) which isexpressed on the surface of lymphocytes and granulocytes. The gene formurine ICAM-1 is disclosed in GenBank under Accession No. X52264 and thegene for the human ICAM-1 homolog, (CD54), is disclosed in Accession No.J03132. In one embodiment, the recombinant vector of the presentinvention contains a foreign nucleic acid sequence encoding at least onemurine ICAM-1 molecule, human homolog, other mammalian homolog orfunctional portion thereof in addition to foreign nucleic acid sequencesencoding two or more additional costimulatory molecules.

The costimulatory molecule leukocyte function antigen 3, murine LFA-3(CD48), and its human homolog LFA-3 (CD58), aglycosyl-phosphatidylinositol-linked glycoprotein, is a member of theCD2 family within the immunoglobulin gene superfamily. The naturalligand of LFA-3 is CD2 (LFA-2) which is expressed on thymocytes, Tcells, B cells and NK cells. The gene for murine LFA-3 is disclosed inGenBank under Accession No. X53526 and the gene for the human homolog isdisclosed in Accession No. Y00636.

The T cell antigen 4-1BBL is a costimulatory molecule that relayscostimulatory signals in antigen-stimulated primary T cell cultures andin lectin-driven activation of thymocytes (Hurtado, J. C. et al J.Immunol. 158(6):2600-2609, 1997). 4-1BBL belongs to the tumor necrosisfactor receptor superfamily, a group of cysteine-rich cell surfacemolecules (Vinay, D. S. et al., Seminars in Immunology 1998, Vol. 10,pp. 481-489). The gene for the murine 4-1BBL is disclosed in GenBankunder Accession No. U02567. The gene for the human homolog, hu4-1BBL isdisclosed in GenBank under Accession No. U03397.

OX-40L is a type II membrane protein with limited homology to TNF and isstimulatory to OX40′ T cells in vitro. The murine and human OX40L cDNAshave 68% homology at the nucleotide level and 46% at the amino acidlevel. Human OX-40L stimulates human T cells exclusively, while murineOX-40L stimulates both human and mouse T cells. APC express OX-40L andcan transmit the OX-40L: OX40R signal during presentation of antigen toCD4 T cells. OX-40L signaling is important for differentiation of humandendritic cells and leads to increased production of IL-12, TNF-α,IL-1B, and IL-6. (Weinberg, A. D. et al 1998 Seminars in Immunology,Vol. 10:471-480). OX-40L is a potent costimulatory molecule forsustaining primary CD4⁺ T cell responses, used in combination with B7-1(Gramaglia, I. et al 1998 J. Immunology, Vol. 161:6510-7.

Vectors having utility in the present invention are capable of causingexpression of at least three or more foreign genes, preferably five ormore foreign genes. Vectors having utility in the present inventioninclude any vector capable of causing functional expression of at leastthree foreign costimulatory molecules gene products in a host cell. Inaddition to the genes encoding at least three costimulatory molecules,the vector is also capable of causing the expression of at least oneforeign gene encoding at least one target antigen or immunologicalepitope thereof as well as a selectable marker.

Vectors of the present invention include but are not limited tobacterial vectors such as Salmonella, viral vectors, nucleic acid basedvectors and the like. Viral vectors include but are not limited topoxvirus, Herpes virus, adenovirus, alphavirus, retrovirus,picornavirus, iridovirus, and the like. Poxviruses having utility in thepresent invention include replicating and non-replicating vectors. Suchpoxviruses include but are not limited to orthopox such as vaccinia,raccoon pox, rabbit pox and the like, avipox, suipox, capripox and thelike. Poxviruses may be selected from the group consisting ofvaccinia-Copenhagen, vaccinia-Wyeth strain, vaccinia-MVA strain, NYVAC,fowlpox, TROVAC, canarypox, ALVAC, swinepox, and the like. In oneembodiment, the recombinant vector is a vaccinia virus. In anotherembodiment, the recombinant vector is fowlpox.

A preferred vector of the present invention is a recombinant virus,preferably a poxvirus. The recombinant poxviruses having utility in thepresent invention have a number of attributes, including (i) efficientdelivery of genes to multiple cell types, including APC and tumor cells;(ii) high levels of protein expression; (iii) optimal presentation ofantigens to the immune system; (iv) the ability to elicit cell-mediatedimmune responses as well as antibody responses; (v) transient, ratherthan permanent, genetic modification of cells, and (vi) the ability touse combinations of poxviruses from different genera, as they are notimmunologically cross-reactive. Parental poxviruses useful inconstructing the recombinant poxvirus of the present invention includebut are not limited to orthopox virus such as replicating vaccinia virus(Perkus et al Science 229:981-984, 1985; Kaufman et al Int. J. Cancer48:900-907, 1991, Moss Science 252:1662, 1991), highly attenuatedvaccinia viruses such as MVA, modified vaccinia Ankara (Sutter and Moss,Proc. Nat'l Acad. Sci. U.S.A. 89:10847-10851; Sutter et al Virology1994), vaccinia-Copenhagen and NYVAC: avipoxviruses (15) such as fowlpoxvirus (15), canary poxviruses, such as ALVAC and the like (Baxby andPaoletti, Vaccine 10:8-9, 1992; Rinns, M. M. et al (Eds) RecombinantPoxviruses CRC Press, Inc, Boca Raton 1992; Paoletti, E. Proc. Nat'lAcad. Sci. USA 93:11349-11353, 1996), and suipoxvirus, capripoxvirus andthe like.

In one embodiment, the parental poxvirus is a vaccinia virus. In aparticular embodiment, the vaccinia virus is a Wyeth strain orderivative thereof. A derivative of the Wyeth strain includes but is notlimited to vTBC33 which lacks a functional K1L gene and the like. In yetanother embodiment, the virus is Dry-Vax available as a smallpox vaccinefrom the Centers for Disease Control, Atlanta, Ga. In anotherembodiment, the parental poxvirus is a strain of fowlpox, for examplePOXVAC-TC (Schering-Plough Corporation), and the like.

The recombinant vector of the present invention is able to infect,transfect or transduce host cells in a host. The host includes but isnot limited to mammals, birds, fish and the like. The host cells are anycell amenable to infection, transfection or transduction by therecombinant vector and capable of expressing the foreign genes from therecombinant vector at functional levels. The host cells include but arenot limited to professional APC and antigen presenting precursor cellssuch as monocytes, macrophages, DC, Langerhans cells and the like. Therecombinant vector of the present invention may also infect tumor cellsor other cell types such as fibroblasts or muscle cells. Infection ofthe host cells allows expression of each foreign, exogenouscostimulatory molecule and expression of the foreign nucleic acidsequence encoding target antigen(s) if present in the recombinantvector. The host cells express, or are engineered to express, theappropriate MHC (HLA) Class I or II molecules for appropriate antigenicpresentation to CD4⁺ and/or CD8⁺ T cells. As such virtually anymammalian cell may be engineered to become an appropriate antigenpresenting cell expressing multiple costimulatory molecules.

The recombinant vector of the present invention comprises at least oneexpression control element operably linked to the nucleic acid sequence.The expression control elements are inserted in the vector to controland regulate the expression of the nucleic acid sequence (Ausubel et al,1987, in “Current Protocols in Molecular Biology, John Wiley and Sons,New York, N.Y.). Expression control elements are known in the art andinclude promoters. Promoters useful in the present invention arepoxyiral promoters as are known in the art which include but are notlimited to 30K, I3, sE/L, 7.5K, 40K, C1 and the like. The nucleic acidsequence of the 30K promoter is disclosed in GenBank Accession No.M35027 at base numbers 28,012 through 28,423 (antisense). The nucleicacid sequence of 13 is disclosed in GenBank Accession No. J03399 at basenumbers 1100 through 1301 (antisense). The nucleic acid sequence of the7.5K promoter is disclosed in GenBank Accession No. M35027 at basenumbers 186550 through 186680. The nucleic acid sequence of the 40Kpromoter is disclosed in GenBank Accession No. M13209 at base numbers9700 through 9858 (antisense). The nucleic acid sequence of the C1promoter is disclosed in GenBank Accession No. M59027 at base numbers 1through 242 and in U.S. Pat. No. 5,093,258. The sequence of the sE/Lpromoter is disclosed in Reference 16. Other poxvirus promoters may beused, such as, those described by Davison and Moss (J. Mol. Biol.210:749-769, (1989). Any of these promoters can be synthesized by usingstandard methods in the art. The selection of an appropriate promoter isbased on its timing and level of expression. Early or early/latepromoters are preferred. In a preferred embodiment, the promoter orcombination of promoters utilized allow for optimal expression of eachcostimulatory molecule in an infected host to provide a synergisticimmune response. In a preferred embodiment, each foreign gene encoding acostimulatory molecule is controlled by a separate and distinctpromoter.

In the case of nucleic acid-based vectors, the constructs may be eithernucleic acid (DNA or RNA) or associated with/or encapsulated in a lipidcarrier. Optionally, the lipid carrier molecule and/or construct mayprovide targeting and/or expression in a particular target cell type ortypes. Naked DNA vectors may be prepared by methods described in U.S.Pat. No. 5,827,703. For the transcriptional initiation region, orpromoter element, any region may be used with the proviso that itprovides the desired level of transcription of the DNA sequence ofinterest. The transcriptional initiation region may be native to orhomologous to the host cell and/or to the DNA to be transcribed, orforeign or heterologous to the host cell and/or the DNA sequence to betranscribed. Efficient promoter elements for transcription initiation ofnaked DNA include but are not limited to the SV40 (simian virus 40)early promoter, the RSV (Rous sarcoma virus) promoter, the adenovirusmajor late promoter, the human CMV (cytomegalovirus) immediate early Ipromoter, and the like. Nucleic acid-based vectors may be delivered to ahost using a syringe, a catheter, or a needle-free injection device suchas a gene gun.

In an embodiment of the invention, a recombinant vector is providedcomprising a foreign nucleic acid sequence encoding a firstcostimulatory molecule or functional portion thereof under control of afirst promoter, a foreign nucleic acid sequence encoding a secondcostimulatory molecule or functional portion thereof under control of asecond promoter, and a foreign nucleic acid sequence encoding a thirdcostimulatory molecule or functional portion thereof under control of athird promoter. The recombinant vector may further provide a foreignnucleic acid sequence encoding a target antigen or immunological portionthereof under control of a fourth promoter.

In one embodiment of the present invention, a recombinant poxvirus isprovided comprising a nucleic acid sequence encoding LFA-3 or functionalportion thereof under control of a 30K poxyiral promoter, a nucleic acidsequence encoding ICAM-1 or portion thereof under control of an 13poxyiral promoter, and a nucleic acid sequence encoding B7.1 or portionthereof under control of an sE/L poxyiral promoter. One example of sucha recombinant poxvirus construct is vaccinia vT171 as depicted in FIG.11A. The recombinant poxvirus may further provide a nucleic acidsequence encoding a tumor associated antigen or immunological portionthereof. One embodiment of the invention is recombinant vaccinia vT172as depicted in FIG. 4C.

In another embodiment of the present invention, a recombinant poxvirusis provided comprising a nucleic acid sequence encoding B7.1 undercontrol of a sE/L poxyiral promoter, a nucleic acid sequence encodingLFA-3 or portion thereof under control of the 13 poxyiral promoter, anda nucleic acid sequence encoding ICAM-1 or portion thereof under controlof the 7.5K poxvirus promoter. Optionally the construct furthercomprises a nucleic acid sequence encoding at least one target antigenor immunological epitope thereof and/or a nucleic acid sequence encodinga selectable marker. One embodiment of such a recombinant poxvirusconstruct is vaccinia vT199 as depicted in FIG. 4B containing a lacZgene as the selectable marker.

In an embodiment of the invention a recombinant fowlpox virus comprisesa nucleic acid sequence encoding B7.1 or portion thereof under controlof the sE/L poxyiral promoter, a nucleic acid sequence encoding LFA-3 orportion thereof under control of the 13 poxyiral promoter, and a nucleicacid sequence encoding ICAM-1 or portion thereof under control of the7.5K poxyiral promoter. An example of this embodiment is fowlpox vT222as depicted in FIG. 4A. A recombinant fowlpox virus may further comprisea nucleic acid sequence encoding a target antigen, CEA, under control ofthe 40K poxyiral promoter and a nucleic acid sequence encoding theselectable marker, lacZ under control of the C1 poxyiral promoter. Anexample of this embodiment is fowlpox vT194 as depicted in FIG. 4B.

In another embodiment, a recombinant fowlpox virus comprises a nucleicacid sequence encoding the tumor-associated antigen MUC-1 or portionthereof under the control of the 40K promoter, a nucleic acid sequenceencoding LFA-3 or portion thereof under the control of the 30K promoter,a nucleic acid sequence encoding ICAM-1 or portion thereof under thecontrol of the 13 promoter, and a nucleic acid sequence encoding B7.1 orportion thereof under the control of the sE/L promoter, as depicted inFIG. 14C. The recombinant fowlpox virus may comprise a nucleic acidsequence encoding any tumor-associated antigen or portion thereof andnucleic acid sequences encoding LFA-3, ICAM-1 and B7.1, under thecontrol of a multiplicity of promoters, as depicted in FIG. 4D.

Another embodiment of the present invention is a recombinant vectorcomprising nucleic acid sequences encoding the human homologs of thecostimulatory molecules LFA-3, B7 and ICAM-1. The recombinant vector mayfurther provide the appropriate promoters to allow expression of eachsequence in an infected host cell. One embodiment of the recombinantvector is vT224 depicted in FIG. 9.

The present invention provides plasmid vectors comprising a foreignnucleic acid sequence encoding multiple costimulatory molecules. In oneembodiment, foreign nucleic acid sequences are selected that encode atleast three or more costimulatory molecules selected from the groupconsisting of B7, ICAM-1, LFA-3, 4-1BBL, CD59, CD40, CD70, VCAM-1,OX-40L and the like. In one embodiment of the present invention, plasmidvectors comprising a foreign nucleic acid sequence encoding at least oneB7 costimulatory molecule, a foreign nucleic acid sequence encoding anICAM-1 costimulatory molecule and a foreign nucleic acid sequenceencoding a LFA-3 costimulatory molecule are provided. The plasmidvectors of the present invention further provide at least one promotersequence for controlling the expression of the costimulatory molecules.In a preferred embodiment each nucleic acid sequence encoding acostimulatory molecule is controlled by a separate discrete promotersequence. For use in making a recombinant poxvirus, the plasmid vectorsof the present invention further provide flanking viral nucleic acidsequences from a non-essential region of a poxvirus genome. The flankingviral nucleic acid sequences direct insertion of the foreign sequencesinto a parental poxyiral genome via homologous recombination. Theplasmid vectors of the present invention may further comprise one ormore selectable markers for selection and identification of recombinantprogeny containing the inserted foreign DNA as are known in the artincluding but not limited to the vaccinia K1L host range gene, the E.coli lacZ gene, antibiotic resistance genes, the gene encodingβ-glucuronidase and the like.

In an embodiment, a plasmid vector of the present invention comprises anucleic acid sequence encoding LFA-3 under control of the 30K promoter,a nucleic acid sequence encoding ICAM-1 under control of the I3 promoterand a nucleic acid sequence encoding B7 under control of the sE/Lpromoter, flanked by portions of the Hind III M region of the vacciniagenome. In one embodiment, the plasmid vector is as depicted in FIG. 1as pT5032.

Another embodiment of the plasmid vector of the present inventioncomprises a nucleic acid sequence encoding B7 under control of the sE/Lpromoter, a nucleic acid sequence encoding LFA-3 under control of the 13promoter and a nucleic acid sequence encoding ICAM-1 under control ofthe 7.5K promoter. The plasmid vector may further comprise a lacZ geneor portion thereof driven by a distinct promoter sequence. Thesesequences are flanked by portions of the Hind III J region of thevaccinia genome. A particular embodiment of the plasmid vector isdepicted as pT5047 in FIG. 2.

In another embodiment of the plasmid vector comprises in combinationwith the nucleic acid sequences encoding B7, ICAM-1, and LFA-3, anucleic acid sequence encoding at least one target antigen orimmunological epitope thereof. A promoter is provided for controllingthe expression of the target antigen. A particular embodiment of theplasmid vector is depicted as pT5031 in FIG. 3 and comprises a nucleicacid sequence encoding the target antigen, CEA.

In another particular embodiment the plasmid vector comprises a nucleicacid sequence encoding the tumor associated antigen, CEA, under controlof the 40K promoter, a nucleic acid sequence encoding B7 under controlof the sE/L promoter, a nucleic acid sequence encoding LFA-3 undercontrol of a 13 promoter and a nucleic acid sequence encoding ICAM-1.The plasmid vector may further comprise a lacZ gene under control of aC1 promoter as depicted in FIG. 6 as pT5049. Plasmid pT5049, wasdeposited with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110 on Nov. 13, 1998 as ATCC Accession No.203481 under the terms of the Budapest Treaty.

In yet another embodiment of the plasmid vector, the vector comprises anucleic acid sequence encoding huLFA-3 under control of the 30Kpromoter, a nucleic acid sequence encoding huICAM-1 under control of anI3 promoter and huB7.1 under control of the sE/L promoter. A particularembodiment of the plasmid vector is depicted as pT5064 in FIG. 8, whichwas deposited with the ATCC on Nov. 13, 1998 as ATCC Accession No.203482 under the terms of the Budapest Treaty.

The plasmid vector of the present invention may be provided in kit formfor use in methods of generating recombinant vectors. The kit mayfurther provide a parental virus, and other reagents used in therecombination process.

The present invention further provides methods of generating recombinantpoxviruses comprising nucleic acid sequences encoding multiplecostimulatory molecules. One method of generation of recombinantpoxviruses is accomplished via homologous recombination in vivo betweenparental poxvirus genomic DNA and a plasmid vector that carries theheterologous sequences to be inserted as disclosed in U.S. Pat. No.5,093,258. Plasmid vectors for the insertion of foreign sequences intopoxviruses are constructed by standard methods of recombinant DNAtechnology (36). The plasmid vectors contain one or more chimericforeign genes, each comprising a poxvirus promoter linked to a proteincoding sequence, flanked by viral sequences from a non-essential regionof the poxvirus genome. The plasmid is transfected into cells infectedwith the parental poxvirus using art accepted transfection methods, andrecombination between poxvirus sequences on the plasmid and thecorresponding DNA in the parental viral genome results in the insertioninto the viral genome of the chimeric foreign genes from the plasmid.Recombinant viruses are selected and purified using any of a variety ofselection or screening systems as are known in the art (14). Insertionof the foreign genes into the vaccinia genome is confirmed by polymerasechain reaction (PCR) analysis. Expression of the foreign genes isdemonstrated by Western blot analysis. An alternative method ofgeneration of recombinant poxviruses is accomplished by direct ligation(Pleiderer et al J. Gen. Virol. 76:2957-2962, 1995; Merchlinsky et alVirol. 238:444-451, 1997).

Use of the recombinant vector comprising nucleic acid sequences encodingmultiple costimulatory molecules in combination with a nucleic acidsequence encoding at least one target antigen or epitope thereof isuseful in enhancing an immune response against the target antigen orepitope thereof, and enhance the immune response against cellsexpressing the target antigen or epitope thereof. The magnitude of theimmune response against the target antigen, epitope, or cells expressingtarget antigen obtained using the recombinant vector of the presentinvention is significantly greater than that achieved using systemsemploying a single or a double costimulatory molecule.

The recombinant vector encodes at least three or more costimulatorymolecules in combination with a nucleic acid sequence encoding a targetantigen or immunological epitope thereof for providing a synergisticimmunological response to the target antigen or epitope thereof. In oneembodiment, a recombinant poxvirus provides a nucleic acid sequenceencoding B7, ICAM-1 and LFA-3, along with a nucleic acid sequenceencoding at least one target antigen or immunological epitope thereof.In some instances it may be beneficial to provide more than one nucleicacid sequence to provide multiple target antigens or immunologicalepitopes thereof for the purpose of having a multivalent vaccine.

The target antigen, as used herein, is an antigen or immunologicalepitope on the antigen which is crucial in immune recognition andultimate elimination or control of the disease-causing agent or diseasestate in a mammal. The immune recognition may be cellular and/orhumoral. In the case of intracellular pathogens and cancer, immunerecognition is preferably a T lymphocyte response.

The target antigen may be derived or isolated from a pathogenicmicroorganism such as viruses including HIV, (Korber et al, eds HIVMolecular Immunology Database, Los Alamos National Laboratory, LosAlamos, N. Mex. 1977) influenza, Herpes simplex, human papilloma virus(U.S. Pat. No. 5,719,054), Hepatitis B (U.S. Pat. No. 5,780,036),Hepatitis C (U.S. Pat. No. 5,709,995), EBV, Cytomegalovirus (CMV) andthe like. Target antigen may be derived or isolated from pathogenicbacteria such as from Chlamydia (U.S. Pat. No. 5,869,608), Mycobacteria,Legionella, Meningiococcus; Group A Streptococcus, Salmonella, Listeria,Hemophilus influenzae (U.S. Pat. No. 5,955,596) and the like.

Target antigen may be derived or isolated from pathogenic yeastincluding Aspergillus, invasive Candida (U.S. Pat. No. 5,645,992),Nocardia, Histoplasmosis, Cryptosporidia and the like.

Target antigen may be derived or isolated from a pathogenic protozoanand pathogenic parasites including but not limited to Pneumocystiscarinii, Trypanosoma, Leishmania (U.S. Pat. No. 5,965,242), Plasmodium(U.S. Pat. No. 5,589,343) and Toxoplasma gondii.

Target antigen includes an antigen associated with a preneoplastic orhyperplastic state. Target antigen may also be associated with, orcausative of cancer. Such target antigen may be tumor specific antigen,tumor associated antigen (TAA) or tissue specific antigen, epitopethereof, and epitope agonist thereof. Such target antigens include butare not limited to carcinoembryonic antigen (CEA) and epitopes thereofsuch as CAP-1, CAP-1-6D (46) and the like (GenBank Accession No.M29540), MART-1 (Kawakami et al, J. Exp. Med. 180:347-352, 1994), MAGE-1(U.S. Pat. No. 5,750,395), MAGE-3, GAGE (U.S. Pat. No. 5,648,226),GP-100 (Kawakami et al Proc. Nat'l Acad. Sci. USA 91:6458-6462, 1992),MUC-1, MUC-2, point mutated ras oncogene, normal and point mutated p53oncogenes (Hollstein et al Nucleic Acids Res. 22:3551-3555, 1994), PSMA(Israeli et al Cancer Res. 53:227-230, 1993), tyrosinase (Kwon et alPNAS 84:7473-7477, 1987, TRP-1 (gp75) (Cohen et al Nucleic Acid Res.18:2807-2808, 1990; U.S. Pat. No. 5,840,839), NY-ESO-1 (Chen et al PNAS94: 1914-1918, 1997), TRP-2 (Jackson et al EMBO J, 11:527-535, 1992),TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2, (U.S. Pat. No. 5,550,214),BRC-I, BRC-II, bcr-abl, pax3-fkhr, ews-fli-1, modifications of TAAs andtissue specific antigen, splice variants of TAAs, epitope agonists, andthe like. Other TAAs may be identified, isolated and cloned by methodsknown in the art such as those disclosed in U.S. Pat. No. 4,514,506.Target antigen may also include one or more growth factors and splicevariants of each.

Possible human tumor antigens and tissue specific antigens as well asimmunological epitopes thereof for targeting using the present inventioninclude but are not limited to those exemplified in Table 1.

TABLE 1 Antigens and Epitopes Recognized by T Cells Target RestrictionImmunological antigens element Peptide epitope SEQ. ID No. Human targettumor antigens reconized by T cells gp 100 HLA-A2 KTWGQYWZY HLA-A2ITDQVPPSV 2 HLA-A2 YLEPGPVTA 3 HLA-A2 LLDGTATLRL 4 HLA-A2 VLYRYGSFSV 5MART1-/Melan A HLA-A2 AAGIGILTV 6 HLA-A2 ILTVILGVL 7 TRP-1 (GP75)HLA-A31 MSLQRQFLR 8 Tyrosinase HLA-A2 MLLAVLYCL 9 HLA-A2 YMNGTMSQV 10HLA-B44 SEIWRDIDF 11 HLA-A24 AFLPWHRLF 12 HLA-DR4 QNILLSNAPLGPQFP 13HLA-DR4 SYLQDSDPDSFQD 14 MAGE-1 HLA-A1 EADPTGHSY 15 HLA-Cw16 SAYGEPRKL16 MAGE-3 HLA-A1 EVDPIGHLY 17 HLA-A2 FLWGPRALV 18 BAGE HLA-Cw16AARAVFLAL 19 GAGE-1,2 HLA-Cw6 YRPRPRRY 20 N-acetylglucos- HLA-A2VLPDVFIRC 21 aminyltransferase-V p15 HLA-A24 AYGLDFYIL 22 CEAYLSGANLNL(CAP1) 23 YLSGADLNL(CAP1-6D) 24 37 β-catenin HLA-A24 SYLDSGIHF25 MUM-1 HLA-B44 EEKLIVVLF 26 CDK4 HLA-A2 ACDPHSGHFV 27 HER-2/neu HLA-A2IlSAVVGIL 28 (Breast and ovarian HLA-A2 KIFGSLAFL 29 carcinoma) Humanpapillomavirus- HLA-A2 YMLDLQPETT 30 E6, E7 (cervical carcinoma) MUC-INon-MHC restricted PDTRPAPGSTAPPAHGVTSA 31 (Breast, ovarian and MHCrestricted (and portions thereof) pancreatic carcinoma) PSA A2, A3FLTPKKLQCVDLHVISNDVCA- 32 QVHPQKVTK FLTPKKLQCV 33 KLQCVDLHV 34VISNDVCAQV 35 QVHPQKVTK 36

For organisms which contain a DNA genome, a gene encoding a targetantigen or immunological epitope thereof of interest is isolated fromthe genomic DNA. For organisms with RNA genomes, the desired gene may beisolated from cDNA copies of the genome. If restriction maps of thegenome are available, the DNA fragment that contains the gene ofinterest is cleaved by restriction endonuclease digestion by methodsroutine in the art. In instances where the desired gene has beenpreviously cloned, the genes may be readily obtained from the availableclones. Alternatively, if the DNA sequence of the gene is known, thegene can be synthesized by any of the conventional techniques forsynthesis of deoxyribonucleic acids.

Genes encoding an antigen of interest can be amplified by cloning thegene into a bacterial host. For this purpose, various prokaryoticcloning vectors can be used. Examples are plasmids pBR322, pUC andpEMBL.

The genes encoding at least one target antigen or immunological epitopethereof can be prepared for insertion into the plasmid vectors designedfor recombination with a virus by standard techniques. In general, thecloned genes can be excised from the prokaryotic cloning vector byrestriction enzyme digestion. In most cases, the excised fragment willcontain the entire coding region of the gene. The DNA fragment carryingthe cloned gene can be modified as needed, for example, to make the endsof the fragment compatible with the insertion sites of the DNA vectorsused for recombination with a virus, then purified prior to insertioninto the vectors at restriction endonuclease cleavage sites (cloningsites) as described herein.

Diseases may be treated or prevented by use of the present invention andinclude diseases caused by viruses, bacteria, yeast, parasites,protozoans, cancer cells and the like. The recombinant vector comprisingmultiple costimulatory molecules may be used as a generalized immuneenhancer and as such has utility in treating diseases of no knownetiological cause.

Preneoplastic or hyperplastic states which may be treated or preventedusing a recombinant vector of the present invention include but are notlimited to preneoplastic or hyperplastic states such as colon polyps,Crohn's disease, ulcerative colitis, breast lesions and the like.

Cancers which may be treated using the recombinant vector of the presentinvention include but are not limited to primary or metastatic melanoma,adenocarcinoma, squamous cell carcinoma, adenosquarnous cell carcinoma,thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkinslymphoma, Hodgkins lymphoma, leukemias, uterine cancer, breast cancer,prostate cancer, ovarian cancer, pancreatic cancer, colon cancer,multiple myeloma, neuroblastoma, NPC, bladder cancer, cervical cancerand the like.

The present invention provides a pharmaceutical composition comprising arecombinant vector comprising foreign genes encoding multiplecostimulatory molecules in a pharmaceutically acceptable carrier. Atleast three genes encoding a costimulatory molecule form part of therecombinant vector and may be selected from the group of genes encodingB7, ICAM-1, LFA-3, 4-1BBL, CD59, CD40, CD70, VCAM-1, OX-40L and thelike. The recombinant vector may further comprise a nucleic acidsequence encoding at least one target antigen or immunological epitopethereof. In another embodiment, the pharmaceutical composition comprisesa first recombinant vector comprising foreign genes encoding multiplecostimulatory molecules, a second recombinant vector comprising nucleicacid sequences encoding at least one target antigen or immunologicalepitope thereof and a pharmaceutically acceptable carrier.Administration of the pharmaceutical composition provides host cellswith the foreign genes encoding multiple costimulatory molecules.

In one embodiment, a pharmaceutical composition comprises a recombinantpoxvirus containing foreign genes encoding multiple costimulatorymolecules in a pharmaceutically acceptable carrier. The recombinantpoxvirus may further comprise a nucleic acid sequence encoding at leastone target antigen or immunological epitope thereof or alternatively, asecond recombinant poxvirus may be provided encoding at least one targetantigen or immunological epitope thereof.

The present invention provides a pharmaceutical composition comprising arecombinant poxvirus comprising a nucleic acid sequence encoding B7.1 toB7.2, a nucleic acid sequence encoding ICAM-1, and a nucleic acidsequence encoding LFA-3 and a pharmaceutically acceptable carrier. Inaddition to the B7, ICAM-1, LFA-3 construct, the recombinant poxvirus ofthe pharmaceutical composition may additionally comprise a nucleic acidsequence encoding at least one target antigen or immunological epitopethereof or the nucleic acid sequence encoding at least one targetantigen or immunological epitope thereof may be provided in thecomposition by a second recombinant poxvirus.

The pharmaceutical composition may also comprise exogenously addedimmunostimulatory molecules as are known in the art including thecostimulatory molecules B7, ICAM-1, LFA-3, 4-1BBL, CD59, CD40, CD70,VCAM-1, OX40L and the like and/or cytokines and chemokines including butnot limited to IL2, GM-CSF, TNFα, IFNγ, IL-12, RANTES, MIP-1α, Flt-3L(U.S. Pat. Nos. 5,554,512; 5,843,423) and the like for additionalsynergy or enhancement of an immune response. The cytokines andchemokines themselves may be provided in the composition or,alternatively, the cytokines and chemokines may be provided by arecombinant viral vector encoding the cytokine or chemokine.

The present invention also encompasses methods of treatment orprevention of a disease caused by pathogenic microorganisms or by cancerdisclosed herein.

In the method of treatment, the administration of the recombinant vectorof the invention may be for either “prophylactic” or “therapeutic”purpose. When provided prophylactically, the recombinant vector of thepresent invention is provided in advance of any symptom. Theprophylactic administration of the recombinant vector serves to preventor ameliorate any subsequent infection or disease. When providedtherapeutically, the recombinant vector is provided at or after theonset of a symptom of infection or disease. Thus the present inventionmay be provided either prior to the anticipated exposure to adisease-causing agent or disease state or after the initiation of theinfection or disease.

The term “unit dose” as it pertains to the inoculum refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of recombinant vector calculated toproduce the desired immunogenic effect in association with the requireddiluent. The specifications for the novel unit dose of an inoculum ofthis invention are dictated by and are dependent upon the uniquecharacteristics of the recombinant virus and the particular immunologiceffect to be achieved.

The inoculum is typically prepared as a solution in tolerable(acceptable) diluent such as saline, phosphate-buffered saline or otherphysiologically tolerable diluent and the like to form an aqueouspharmaceutical composition.

The route of inoculation may be scarification, intravenous (I.V.),intramuscular (I.M.), subcutaneous (S.C.), intradermal (I.D.),intraperitoneal (I.P.), intratumor and the like, which results ineliciting a protective response against the disease causing agent. Thedose is administered at least once. Subsequent doses may be administeredas indicated.

In one embodiment, heterologous prime-boost regimens are employed. Inone example, the host is immunized at least once with a first vectorsuch as a nucleic acid-based vector. Subsequent immunizations areperformed with a poxvirus vector. In another example, the host is firstimmunized with a first poxvirus vector and then with a second poxvirusvector of a different genus.

In providing a mammal with the recombinant vector of the presentinvention, preferably a human, the dosage of administered recombinantvector will vary depending upon such factors as the mammal's age,weight, height, sex, general medical condition, previous medicalhistory, disease progression, tumor burden and the like.

In general, it is desirable to provide the recipient with a dosage ofrecombinant virus in the range of about 10⁵ to about 10¹⁰ plaque formingunits, although a lower or higher dose may be administered.

The genetic definition of tumor-associated and tumor-specific antigensallows for the development of targeted antigen-specific vaccines forcancer therapy. Insertion of a tumor antigen gene in the genome ofrecombinant pox viruses in combination with genes encoding multiplecostimulatory molecules is a powerful system to elicit a specific immuneresponse in terms of prevention in individuals with an increased risk ofcancer development (preventive immunization), to shrink tumors prior tosurgery, to prevent disease recurrence after primary surgery(anti-metastatic vaccination), or to expand the number of cytotoxiclymphocytes (CTL) in vivo, thus improving their effectiveness ineradication of diffuse tumors (treatment of established disease).Recombinant viruses of the present invention can elicit an immuneresponse ex vivo in autologous lymphocytes (CD8⁺), either cytotoxic Tlymphocytes and/or CD4⁺ helper T cells or NK cells prior to beingtransferred back to the tumor bearing patient (adoptive immunotherapy).

In cancer treatments, the recombinant vectors can be introduced into amammal either prior to any evidence of cancers such as an adenocarcinomaor to mediate regression of the disease in a mammal afflicted with acancer such as adenocarcinoma.

Depending on the disease or condition to be treated and the method oftreatment, the recombinant vector may or may not comprise a nucleic acidsequence encoding a target antigen or immunological epitope thereof inaddition to the genes encoding multiple costimulatory molecules. Thetarget antigen or immunological epitope thereof may be providedendogenously by the host cell infected with the recombinant vector as,for instance, a tumor cell may endogenously express a tumor associatedantigen or epitope thereof and may not require the addition of a foreigngene encoding an exogenous tumor associated antigen. In the case inwhich a tumor associated antigen is absent, not expressed or expressedat low levels in a host cell, a foreign gene encoding an exogenous tumorassociated antigen may be provided. Further, genes encoding severaldifferent tumor associated antigens may be provided. The foreign geneencoding an exogenous tumor associated antigen may be provided by thesame recombinant vector comprising genes encoding multiple costimulatorymolecules or may be provided by a second recombinant vector in anadmixture with the first recombinant vector.

Examples of methods for administering the recombinant vector intomammals include, but are not limited to, exposure of tumor cells to therecombinant virus ex vivo, or injection of the recombinant vector intothe affected host by intravenous, S.C., I.D. or I.M. administration ofthe virus. Alternatively the recombinant vector or combination ofrecombinant vectors may be administered locally by direct injection intothe cancerous lesion or tumor or topical application in apharmaceutically acceptable carrier. The quantity of recombinant vectorcarrying the nucleic acid sequence of one or more tumor associatedantigens (TAAs) in combination with nucleic acid sequences encodingmultiple costimulatory molecules to be administered is based on thetiter of virus particles. A preferred range of the immunogen to beadministered is 10⁵ to 10¹⁰ virus particles per mammal, preferably ahuman. If the mammal to be immunized is already afflicted with cancer ormetastatic cancer, the vaccine can be administered in conjunction withother therapeutic treatments.

In one method of treatment, autologous cytotoxic lymphocytes or tumorinfiltrating lymphocytes may be obtained from blood, lymph nodes, tumorand the like from a patient with cancer. The lymphocytes are grown inculture and target antigen-specific lymphocytes are expanded byculturing in the presence of specific target antigen and either antigenpresenting cells expressing multiple foreign costimulatory molecules ortarget antigen pulsed APCs of the present invention. The targetantigen-specific lymphocytes are then reinfused back into the patient.

After immunization the efficacy of the vaccine can be assessed byproduction of antibodies or immune cells that recognize the antigen, asassessed by specific lytic activity or specific cytokine production orby tumor regression. One skilled in the art would know the conventionalmethods to assess the aforementioned parameters.

The present invention encompasses methods of enhancing antigen-specificT-cell responses by administration of an effective amount of arecombinant vector encoding multiple foreign costimulatory molecules anda target antigen into a mammal alone, or infecting a target cell withthe vector, target antigen or immunological epitope thereof. In oneembodiment of the method, a recombinant vector encoding at least onemolecule from the B7 family, ICAM-1 and LFA-3 is administered alone, oradmixed with a target cell, target antigen or immunological epitopethereof. This immunization approach augments or enhances immuneresponses generated by the target antigen, providing a synergisticresponse compared to the use of single or double costimulatorymolecules. The method of administering a recombinant vector containinggenes encoding multiple costimulatory molecules results in increasedtarget antigen-specific lymphoproliferation, enhanced cytolyticactivity, enhanced cytokine secretion and longer lasting immunity to thetarget antigen as compared to the use of recombinant vector encoding asingle or double costimulatory molecule. The recombinant vector mayfurther comprise a nucleic acid-sequence encoding at least one targetantigen or immunological epitope thereof for synergistic enhancement oftarget-antigen-specific immune responses. Alternatively, the nucleicacid sequence encoding at least one target antigen or immunologicalepitope thereof is provided by a second recombinant vector, distinctfrom the vector encoding the multiple costimulatory molecules. In oneembodiment of the method of enhancing antigen-specific T-cell responses,mammals, preferably humans, are immunized with an rV-HIV-1epitope/B7-1/ICAM-1/LFA-3 construct. The efficacy of the treatment maybe monitored in vitro and/or in vivo by determining targetantigen-specific lymphoproliferation, target antigen-specific cytolyticresponse, clinical responses and the like.

The method of enhancing antigen-specific T-cell responses may be usedfor any target antigen or immunological epitope thereof. Of particularinterest are tumor associated antigens, tissue specific antigens andantigens of infectious agents.

In addition to administration of the recombinant vector to the patient,other exogenous immunomodulators or immunostimulatory molecules,chemotherapeutic drugs, antibiotics, antifungal drugs, antiviral drugsand the like alone or in combination thereof may be administereddepending on the condition to be treated. Examples of other exogenouslyadded agents include exogenous IL-2, IL-6, alpha-, beta- orgamma-interferon, GM-CSF, tumor necrosis factor, Flt-3L,cyclophosphamide, cisplatinum, gancyclovir, amphotericin B, 5fluorouracil and the like.

The present invention provides for host cells expressing multiple,exogenous foreign costimulatory molecules in which the molecules areprovided by a recombinant vector having foreign nucleic acid sequencesencoding multiple costimulatory molecules. The host cells may alsoexpress one or more endogenous target antigens or immunological epitopesthereof or may be engineered to express one or more exogenous, foreigntarget antigens or immunological epitopes thereof which may also beprovided by the recombinant vector encoding multiple costimulatorymolecules or by a second recombinant vector.

The host cells of the present invention, with utility in stimulating anantigen-specific immune response may be any cell capable of infectionusing the recombinant virus of the present invention and capable ofexpressing multiple, exogenous costimulatory molecules and may furtherbe genetically engineered to express one or more exogenous targetantigens or immunological epitopes thereof. Such host cells included butare not limited to tumor cells, antigen presenting cells, such as PBMC,dendritic cells, cells of the skin or muscle, and the like. Antigenpresenting cells include, but are not limited to, monocytes,macrophages, dendritic cells, progenitor dendritic cells, Langerhanscells, splenocytes, B-cells, tumor cells, muscle cells, epithelial cellsand the like.

In one embodiment, the host cells are tumor cells in which the tumorcells are exposed to the recombinant vector in situ or in vitro to causeexpression of multiple foreign or exogenous costimulatory molecules onthe tumor cells. The tumor cells may express an endogenous targetantigen or the tumor cells may be further genetically engineered toexpress a target antigen such as TAA or immunological epitope thereof.Tumor cells expressing both the TAA along with multipleimmunostimulatory molecules are administered to a mammal in an effectiveamount to result in tumor reduction or elimination in the mammalafflicted with a cancer.

The present invention also provides progenitor dendritic cells,dendritic cells (DC), DC subpopulations, and derivatives thereofoverexpressing multiple costimulatory molecules in which multiplecostimulatory molecules are exogenously provided by a recombinant vectorhaving nucleic acid sequences encoding multiple costimulatory molecules.The progenitor DC and DC of the present invention express higher levelsof costimulatory molecules, than levels endogenously expressed by anontreated progenitor DC or DC. The APCs such as progenitor dendriticcells and dendritic cells may also express one or more endogenous targetantigens or immunological epiltopes thereof or exogenous target antigenmay also be provided by the recombinant vector encoding multiplecostimulatory molecules or by a second recombinant vector. The presentinvention further provides methods of using the multiple costimulatorymolecule-overexpressing APCs, such as multiple costimulatorymolecule-overexpressing progenitor dendritic cells and multiplecostimulatory molecule-overexpressing dendritic cells in activating Tcells in vivo or in vitro for vaccination and immunotherapeuticresponses against one or more target cells, target antigens andimmunological epitopes thereof.

The APCs such as progenitor dendritic cells, dendritic cells, DCsubpopulations and derivatives thereof isolated from a source areinfected, transfected or transduced with a recombinant vector comprisingexogenous genes encoding at least three costimulatory molecules for atime period sufficient to allow functional overexpression of themultiple costimulatory molecules. Such multiple costimulatorymolecule-overexpressing antigen presenting progenitor dendritic cellsand dendritic cells may also be pulsed or incubated with at least onetarget cell, target cell lysate, target cell membrane, target antigen,or immunological epitope thereof, or with RNA or DNA of at least onetarget cell and administered to a species in an amount sufficient toactivate the relevant T cell responses in vivo. In another embodiment,the antigen presenting progenitor dendritic cells and dendritic cellsadditionally express at least one foreign target antigen orimmunological epitope thereof.

Host cells expressing multiple, exogenous costimulatory molecules may beprovided in a dose of 10³ to 10⁹ cells. Routes of administration thatmay be used include intravenous, subcutaneous, intralymphatic,intratumoral, intradermal, intramuscular, intraperitoneal, intrarectal,intravaginal, intranasal, oral, via bladder instillation, viascarification, and the like.

In one embodiment, the multiple costimulatory molecule-overexpressingantigen presenting progenitor dendritic cells or dendritic cells areexposed to a target cell, target cell lysates, target cell membranes,target antigen or immunological epitope thereof or with DNA or RNA fromat least one target cell in vitro and incubated with primed or unprimedT cells to activate the relevant T cell responses in vitro. Theactivated T cells alone or in combination with the progenitor DC or DCare then administered to a species such as a human for vaccination orimmunotherapy against a target cell, target antigen or immunologicalepitope thereof. In one method of use, the progenitor dendritic cells ordendritic cells are advantageously used to elicit an immunotherapeuticgrowth inhibiting response against cancer cells.

In another embodiment, the multiple costimulatorymolecule-overexpressing antigen-presenting cell, preferably a precursorDC or DC is fused with a target cell expressing a relevant targetantigen or immunological epitope thereof to form a heterokaryon of APCand target cell by methods known in the art (Gong, J. et al Proc. Natl.Acad. Sci. USA 95:6279-6283, 1998). Such a fusion cell or chimericAPC/target antigen cell expresses both multiple costimulatory moleculesand target antigen or immunological epitopes thereof. In a preferredembodiment the target cell is a hyperplastic cell, premalignant ormalignant cell. The chimeric APC/target antigen cell may be used both invivo and in vitro to enhance immune responses of T and B lymphocytes.

Progenitor dendritic cells are obtained from bone marrow, peripheralblood and lymph nodes from a patient. The patient may have beenpreviously vaccinated, or treated with a compound such as Flt-3L toenhance the number of antigen-presenting cells. Dendritic cells areobtained from any tissue such as the epidermis of the skin (Langerhanscells) and lymphoid tissues such as found in the spleen, bone marrow,lymph nodes, and thymus as well as the circulatory system includingblood and lymph (veiled cells). Cord blood is another source ofdendritic cells.

Dendritic cells may be enriched or isolated for use in the presentinvention using methods known in the art such as those described in U.S.Pat. No. 5,788,963. Once the progenitor dendritic cells, dendritic cellsand derivatives thereof are obtained, they are cultured underappropriate culture conditions to expand the cell population and/ormaintain the cells in a state for optimal infection, transfection ortransduction by a recombinant vector and for optimal target antigenuptake, processing and presentation. Particularly advantageous formaintenance of the proper state of maturity of dendritic cells in invitro culture is the presence of both the granulocyte/macrophage colonystimulating factor (GM-CSF) and interleukin 4 (IL-4). Subpopulations ofdendritic cells may be isolated based in adherence and/or degree ofmaturity based on cell surface markers. The phenotype of the progenitorDC, DC and subpopulations thereof are disclosed in Banchereau andSteinman Nature 392:245-252, 1998.

In one embodiment GM-CSF and IL-4 are each provided in a concentrationof about 500 units/ml for a period of about 6 days (41,42). In anotherembodiment, TNFα and/or CD40 is used to cause precursor DC or DC tomature.

The progenitor dendritic cells or dendritic cells may be obtained fromthe individual to be treated and as such are autologous in terms ofrelevant HLA antigens or the cells may be obtained from an individualwhose relevant HLA antigens (both class I and II, e.g. HLA-A, B, C andDR) match the individual that is to be treated. Alternatively, theprogenitor dendritic cell is engineered to express the appropriate,relevant HLA antigens of the individual receiving treatment.

The progenitor dendritic cells and dendritic cells may be furthergenetically modified to extend their lifespan by such methods asEBV-transformation as disclosed in U.S. Pat. No. 5,788,963.

The dendritic cells and precursors thereof may be provided in the formof a pharmaceutical composition in a physiologically acceptable medium.The composition may further comprise a target cell, target cell lysate,target cell membrane, target antigen or immunological epitope thereof.The composition may additionally comprise cytokines and/or chemokinessuch as IL-4 and GM-CSF for additional synergistic enhancement of animmune response.

In another embodiment, the APC of the present invention overexpressingmultiple costimulatory molecules is useful in methods of evaluatingefficacy of a vaccine by determination of antigen-specific lymphocyteproliferation and function. In such a method, lymphocytes are recoveredfrom an individual who has been vaccinated with a target cell lysate,target cell membrane, target antigen or immunological epitope thereof.The lymphocytes are cultured in vitro with an APC of the presentinvention in the presence of the target cell, target cell lysate, targetcell membrane, target antigen or immunological epitope thereof and anenhancement of antigen-specific lymphocyte numbers and functionsdetermined by methods known in the art. An enhancement in numbers and/orfunctions is indicative of efficacy of the vaccine. The method isparticularly useful in determining efficacy of peptide vaccines instimulating an appropriate immune response.

In another embodiment, the APCs of the present invention expressingexogenous multiple costimulatory molecules are useful in a method ofscreening for novel immunogenic peptides from a multiplicity ofpeptides. In the method of screening, antigen presenting cells infectedwith a recombinant vector encoding multiple costimulatory molecules orfunctional portions thereof are pulsed with a multiplicity of peptidesto form a peptide-pulsed antigen presenting cell. The peptide-pulsedantigen presenting cell is incubated with lymphoid cells and theimmunoreactivity of the lymphoid cells measured. An enhancement ofimmunoreactivity of the lymphoid cells in the presence of thepeptide-pulsed APC is indicative of an antigen specific response to thepeptide. The peptide eliciting the enhanced response can be identifiedby eluting from tumor, by analysis of HLA binding, etc. The source ofthe multiplicity of peptides may be a combinatorial library whichexpresses a multiplicity of random peptides. The enhancedimmunoreactivity may be a humoral or cell-mediated immune response andmay be measured using standard techniques known in the art such asantigen-induced proliferation, cytotoxicity, antibody secretion, signaltransduction, and the like. The novel peptides identified may be used asimmunogens, vaccines or diagnostic agents. The proteins that contain thepeptides may be identified by subtraction libraries and differentialdisplay gene technologies.

The recombinant vectors of the present invention as well as host cellsinfected, transfected or induced by the recombinant vector of thepresent invention are useful in methods of stimulating an enhancedhumoral response both in vivo and in vitro. Such an enhanced humoralresponse may be monoclonal or polyclonal in nature. The enhancement ofhumoral responses using multiple costimulatory molecules is synergisticas compared to a humoral response using a single or double costimulatorymolecule. The synergistic enhancement of a humoral response may bedetermined by increased proliferation and/or cytokine secretion by CD4⁺T cells, increased proliferation or antibody production by B cells,increased antibody dependent cellular toxicity (ADCC), increasedcomplement-mediated lysis, and the like. Antibody elicited using therecombinant vectors of the present invention or using host cellsinfected, transfected or induced by the recombinant vector of thepresent invention are expected to be higher affinity and/or avidity andhigher titer than antibody elicited by standard methods. The antibodyelicited by methods using the recombinant vector may recognizeimmunodominant target epitopes or nondominant target epitopes.

This invention further comprises an antibody or antibodies elicited byimmunization with the recombinant vector of the present invention. Theantibody has specificity for and reacts or binds with the target antigenor immunological epitope thereof of interest. In this embodiment of theinvention the antibodies are monoclonal or polyclonal in origin.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules or those portions of animmunoglobulin molecule that contain the antigen binding site, includingthose portions of immunoglobulin molecules known in the art as F(ab),F(ab′), F(ab)₂ and F(v). Polyclonal or monoclonal antibodies may beproduced by methods known in the art. (Kohler and Milstein (1975) Nature256, 495-497; Campbell “Monoclonal Antibody Technology, the Productionand Characterization of Rodent and Human Hybridomas” in Burdon et al.(eds.) (1985) “Laboratory Techniques in Biochemistry and MolecularBiology,” Volume 13, Elsevier Science Publishers, Amsterdam). Theantibodies or antigen binding fragments may also be produced by geneticengineering. The technology for expression of both heavy and light chaingenes in E. coli is the subject of the PCT patent applications:publication number WO 901443, WO 901443 and WO 9014424 and in Huse etal. (1989) Science 246:1275-1281.

In one embodiment the antibodies of this invention are used inimmunoassays to detect the novel antigen of interest in biologicalsamples.

In one embodiment, the antibodies of this invention generated byimmunization with a recombinant vaccinia virus expressing a TAA andexpressing B7-1, ICAM-1 and LFA-3 are used to assess the presence of thea TAA from a tissue biopsy of a mammal afflicted with a cancerexpressing TAA using immunocytochemistry. Such assessment of thedelineation of the a TAA antigen in diseased tissue can be used toprognose the progression of the disease in a mammal afflicted with thedisease or the efficacy of immunotherapy. In this embodiment, examplesof TAAs include but are not limited to CEA, PSA, and MUC-1. Conventionalmethods for immunohistochemistry are described in (Harlow and Lane (eds)(1988) In “Antibodies A Laboratory Manual”, Cold Spinning Harbor Press,Cold Spring Harbor, N.Y.; Ausubel et al. (eds) (1987). In CurrentProtocols In Molecular Biology, John Wiley and Sons (New York, N.Y.).

In another embodiment the antibodies of the present invention are usedfor immunotherapy. The antibodies of the present invention may be usedin passive immunotherapy.

In providing a patient with the antibodies or antigen binding fragmentsto a recipient mammal, preferably a human, the dosage of administeredantibodies or antigen binding fragments will vary depending upon suchfactors as the mammal's age, weight, height, sex, general medicalcondition, previous medical condition and the like.

The antibodies or antigen-binding fragments of the present invention areintended to be provided to the recipient subject in an amount sufficientto prevent, lessen or attenuate the severity, extent or duration of thedisease or infection.

Anti-idiotypic antibodies arise normally during the course of immuneresponses, and a portion of the anti-idiotype antibody resembles theepitope that induced the original immune response. In the presentinvention, the immunoglobulin gene or portion thereof of an antibodywhose binding site reflects a target antigen of a disease state, isincorporated into the genome or portion thereof of a virus genome, aloneor in combination with a gene or portion thereof of multipleimmunostimulatory molecules, the resulting recombinant virus is able toelicit cellular and humoral immune response to the antigen.

The description of the specific embodiments will so fully reveal thegeneral nature of the invention that others can readily modify and/oradopt for various purposes such specific embodiments without departingfrom the generic concept, and therefor such adaptations andmodifications are intended to be comprehended within the meaning andrange of equivalents of the disclosed embodiments.

All references and patents referred to are incorporated herein byreference.

EXAMPLE 1 Generation of Recombinant Vaccinia, rV-TRICOM(mu1) No. vT171

The origin of vaccinia parental virus is the New York City Board ofHealth strain and was obtained by Wyeth from the New York City Board ofHealth and passaged in calves to create the Smallpox Vaccine Seed. FlowLaboratories received a lyophilized vial of the Smallpox Vaccine Seed,Lot 3197, Passage 28 from Drs. Chanock and Moss (National Institutes ofHealth). This seed virus was ether-treated and plaque-purified threetimes.

For the generation of rV-TRICOM(mu1), a plasmid vector, designatedpT5032 was constructed to direct insertion of the foreign sequences intothe M2L (30K) gene, which is located in the Hind III M region of thevaccinia genome. The murine LFA-3 gene is under the transcriptionalcontrol of the vaccinia 30K (M2L) promoter (34), the murine ICAM-1 geneis under the control of the vaccinia 13 promoter (18), and the murineB7.1 gene is under the control of the synthetic early/late (sE/L)promoter (32). These foreign sequences are flanked by DNA sequences fromthe Hind III M region of the vaccinia genome (see FIG. 1). Theseflanking sequences include the vaccinia K1L host range gene (33). Aderivative of the Wyeth strain of vaccinia was used as the parentalvirus in the construction of recombinant vaccinia virus. This parentalvirus, designated vTBC33, lacks a functional K1L gene and thus cannotefficiently replicate on rabbit kidney RK₁₃ cells (38). The generationof recombinant vaccinia virus was accomplished via homologousrecombination between vaccinia sequences in the vTBC33 vaccinia genomeand the corresponding sequences in pT5032 in vaccinia-infected RK₁₃cells transfected with pT5032. Recombinant virus, designated vT171, wasselected by growth on RK₁₃ cells (ATCC, CCL 37). Plaques were pickedfrom the cell monolayer and their progeny were further propagated. Tworounds of plaque isolation and replating on RK₁₃ cells resulted in thepurification of the desired recombinant. The genomic structure ofrecombinant vT171 is depicted in FIG. 4A.

EXAMPLE 2 Generation of Recombinant Vaccinia, rV-TRICOM(mu2) No. vT199

For the generation of rV-TRICOM(mu2), a plasmid vector, designatedpT5047, was constructed to direct insertion of the foreign sequencesinto the thymidine kinase (TK) gene, which is located in the Hind III Jregion of the vaccinia genome. The murine B7.1 gene is under the controlof the sE/L promoter, the murine LFA-3 gene is under the transcriptionalcontrol of the 13 promoter, and the murine ICAM-1 gene is under thecontrol of the vaccinia 7.5K promoter (39). In addition, the E. colilacZ gene, under the control of the fowlpox virus C1 promoter (15) isincluded as a screen for recombinant progeny. These foreign sequencesare flanked by DNA sequences from the Hind III J region of the vacciniagenome (see FIG. 2). A plaque-purified isolate from the Wyeth (New YorkCity Board of Health) strain of vaccinia was used as the parental virusfor this recombinant vaccine. The generation of recombinant vacciniavirus was accomplished via homologous recombination between vacciniasequences in the Wyeth vaccinia genome and the corresponding sequencesin pT5047 in vaccinia-infected Hu 143TK-cells (Bacchetti and Graham1977) transfected with pT5047. Recombinant virus was identified usingselection for TK virus in the presence of bromodeoxyuridine (BudR) incombination with a chromogenic assay, performed on viral plaques insitu, that detects expression of the lacZ gene product in the presenceof halogenated indolyl-beta-D-galactoside (Bluo-gal), as describedpreviously (31). Viral plaques expressing lacZ appeared blue against aclear background. Positive plaques, designated vT199, were picked fromthe cell monolayer and their progeny were replated under the selectiveconditions described above. In other recombinant viruses selected andpurified in this manner, the only plaques that appeared under theseselective conditions were blue, and these blue plaques were readilyisolated and purified. However, in the case of vT199, only white plaqueswere observed at the second round of plaque-purification; no blueplaques were apparent. A new set of blue plaques were picked andreplated; again, only white plaques were observed at the second round ofplaque-purification. A final attempt, using yet another set of blueplaques, yielded both blue and white plaques after the second round ofplaque-purification. Blue plaques were selected and replated. Twoadditional rounds of plaque-purification (a total of four rounds)yielded recombinant viruses that were 100% blue. The genomic structureof recombinant vT199 is depicted in FIG. 4B.

EXAMPLE 3 Generation of Recombinant Vaccinia rV-TAA/TRICOM(mu)

For the generation of rV-TAA/TRICOM(mu), a plasmid vector is constructedto direct insertion of the foreign sequences into the vaccinia genome.The TAA gene, the murine LFA-3 gene, the murine ICAM-1 gene, and themurine B7.1 gene are under the control of a multiplicity of promoters.These foreign sequences are flanked by DNA sequences from the vacciniagenome, into which the foreign sequences are to be inserted. Thegeneration of recombinant vaccinia virus is accomplished via homologousrecombination between vaccinia sequences in the vaccinia genome and thecorresponding sequences in the plasmid vector in vaccinia-infected cellstransfected with the plasmid vector. Recombinant plaques are picked fromthe cell monolayer under selective conditions and their progeny arefurther propagated. Additional rounds of plaque isolation and replatingresult in the purification of the desired recombinant virus.

EXAMPLE 4 Generation of Recombinant Vaccinia rV-MUC-1/TRICOM(mu)

For the generation of rV-MUC-1/TRICOM(mu), a plasmid vector isconstructed to direct insertion of the foreign sequences into thevaccinia genome. The MUC-1 gene, the murine LFA-3 gene, the murineICAM-1 gene, and the murine B7.1 gene are under the control of amultiplicity of promoters. These foreign sequences are flanked by DNAsequences from the vaccinia genome into which the foreign sequences areto be inserted. The generation of recombinant vaccinia virus isaccomplished via homologous recombination between vaccinia sequences inthe vaccinia genome and the corresponding sequences in the plasmidvector in vaccinia-infected cells transfected with the plasmid vector.Recombinant plaques are picked from the cell monolayer under selectiveconditions and their progeny are further propagated. Additional roundsof plaque isolation and replating result in the purification of thedesired recombinant virus.

EXAMPLE 5 Generation of Recombinant Vaccinia rV-CEA/TRICOM(mu) No. vT172

For the generation of rV-CEA/TRICOM(mu), a plasmid vector, designatedpT5031, was constructed to direct insertion of the foreign sequencesinto the M2L (30K) gene, which is located in the Hind III M region ofthe vaccinia genome (see FIG. 3). The CEA gene is under the control ofthe 40K promoter (13), the murine LFA-3 gene is under the control of the30K promoter, the murine ICAM-1 gene is under the control of the 13promoter, and the murine B7.1 gene is under the control of the sE/Lpromoter. These foreign sequences are flanked by DNA sequences from theHind III M region of the vaccinia genome, including the vaccinia K1Lhost range gene. vTBC33, described above, was used as the parental virusin the construction of the recombinant vaccinia virus. The generation ofrecombinant vaccinia virus was accomplished via homologous recombinationbetween vaccinia sequences in the vTBC33 vaccinia genome and thecorresponding sequences in pT5031 in vaccinia-infected RK₁₃ cellstransfected with pT5031. Recombinant virus, designated vT172, wasselected by growth on RK₁₃ cells as described above. Plaques were pickedfrom the cell monolayer and their progeny were further propagated. Tworounds of plaque isolation and replating on RK13 cells resulted in thepurification of the desired recombinant. The genomic structure ofrecombinant vT172 is depicted in FIG. 4C.

EXAMPLE 6 Generation of Recombinant Fowlpox, rF-TRICOM(mu) No. vT222

The origin of parental fowlpox virus used for the generation ofrecombinants was plaque-purified from a vial of a USDA-licensed poultryvaccine, POXVAC-TC, which is manufactured by Schering-PloughCorporation. The starting material for the production of POXVAC-TC was avial of Vineland Laboratories' chicken embryo origin Fowlpox vaccine,obtained by Schering-Plough. The virus was passaged twice on thechorioallantoic membrane of chicken eggs to produce a master seed virus.The master seed virus was passaged 27 additional times in chicken embryofibroblasts to prepare the POXVAC-TC master seed. To prepare virusstocks for the generation of POXVAC-TC product lots, the POXVAC-TCmaster seed was passaged twice on chicken embryo fibroblasts. One vialof POXVAC-TC, Serial # 96125, was plaque-purified three times on primarychick embryo dermal cells.

For the generation of rF-TRICOM(mu), a plasmid vector, designatedpT8001, was constructed to direct insertion of the foreign sequencesinto the BamHI J region of the fowlpox genome. The murine B7.1 gene isunder the control of the sE/L promoter, the murine LFA-3 gene is underthe control of the 13 promoter, the murine ICAM-1 gene is under thecontrol of the 7.5K promoter, and the lacZ gene is under the control ofthe C1 promoter. These foreign sequences are flanked by DNA sequencesfrom the BamHI J region of the fowlpox genome (see FIG. 5). Aplaque-purified isolate from the POXVAC-TC (Schering-Plough Corporation)strain of fowlpox was used as the parental virus for this recombinantvaccine. The generation of recombinant fowlpox virus was accomplishedvia homologous recombination between fowlpox sequences in the fowlpoxgenome and the corresponding sequences in pT8001 in fowlpox-infectedprimary chick embryo dermal cells transfected with pT8001. Recombinantvirus was identified using the chromogenic assay for the lacZ geneproduct described above. Viral plaques expressing lacZ appeared blueagainst a clear background. Positive plaques, designated vT222, werepicked from the cell monolayer and their progeny were replated. Sixrounds of plaque isolation and replating in the presence of Bluo-Galresulted in the purification of the desired recombinant. The genomicstructure of recombinant vT222 is depicted in FIG. 7A.

EXAMPLE 7 Generation of Recombinant Fowlpox rF-TAA/TRICOM(mu)

For the generation of rF-TAA/TRICOM(mu), a plasmid vector is constructedto direct insertion of the foreign sequences into the BamHI J region ofthe fowlpox genome. The TAA gene, the murine LFA-3 gene, the murineICAM-1 gene, and the murine B7.1 gene are under the control of amultiplicity of promoters. In addition, the lacZ gene is under thecontrol of the C1 promoter. These foreign sequences are flanked by DNAsequences from the BamHI J region of the fowlpox genome. Aplaque-purified isolate from the POXVAC-TC (Schering-Plough Corporation)strain of fowlpox is used as the parental virus for this recombinantvaccine. The generation of recombinant fowlpox virus is accomplished viahomologous recombination between fowlpox sequences in the fowlpox genomeand the corresponding sequences in the plasmid vector infowlpox-infected primary chick embryo dermal cells transfected with theplasmid vector. Recombinant virus is identified using the chromogenicassay for the lacZ gene product described above. Viral plaquesexpressing lacZ appear blue against a clear background. Positive plaquesare picked from the cell monolayer and their progeny are replated.Additional rounds of plaque isolation and replating in the presence ofBluo-Gal result in the purification of the desired virus.

EXAMPLE 8 Generation of Recombinant Fowlpox rF-MUC-1/TRICOM(mu)

For the generation of rF-MUC-1/TRICOM(mu), a plasmid vector isconstructed to direct insertion of the foreign sequences into the BamHIJ region of the fowlpox genome. The MUC-1 gene, the murine LFA-3 gene,the murine ICAM-1 gene, and the murine B7.1 gene are under the controlof a multiplicity of promoters. In addition, the lacZ gene is under thecontrol of C1 promoter. These foreign sequences are flanked by DNAsequences from the BamHI J region of the fowlpox genome. Aplaque-purified isolate from the POXVAC-TC (Schering-Plough Corporation)strain of fowlpox is used as the parental virus for this recombinantvaccine. The generation of recombinant fowlpox virus is accomplished viahomologous recombination between fowlpox sequences in the fowlpox genomeand the corresponding sequences in the plasmid vector infowlpox-infected primary chick embryo dermal cells transfected with theplasmid vector. Recombinant virus is identified using the chromogenicassay for the lacZ gene product described above. Viral plaquesexpressing lacZ appear blue against a clear background. Positive plaquesare picked from the cell monolayer and their progeny are replated.Additional rounds of plaque isolation and replating in the presence ofBluo-Gal result in the purification of the desired recombinant virus.

EXAMPLE 9 Generation of Recombinant Fowlpox, rF-CEA/TRICOM(mu) No. vT194

For the generation of rF-CEA/TRICOM(mu), a plasmid vector, designatedpT5049, was constructed to direct insertion of the foreign sequencesinto the BamHI J region of the fowlpox genome. The CEA gene is under thecontrol of the vaccinia 40K promoter, the murine B7-1 gene is under thecontrol of the sE/L promoter, the murine LFA-3 gene is under thetranscriptional control of the 13 promoter, the murine ICAM-1 gene isunder the transcriptional control of the vaccinia 7.5K promoter, and thelacZ gene is under the control of the C1 promoter. These foreignsequences are flanked by DNA sequences from the BamHI J region of thefowlpox genome (see FIG. 6). A plaque-purified isolate from thePOXVAC-TC (Schering Corporation) strain of fowlpox was used as theparental virus for this recombinant vaccine. The generation ofrecombinant fowlpox virus was accomplished via homologous recombinationbetween fowlpox sequences in the fowlpox genome and the correspondingsequences in pT5049 in fowlpox-infected primary chick embryo dermalcells transfected with pT5049. Recombinant virus was identified usingthe chromogenic assay for the lacZ gene product described above. Viralplaques expressing lacZ appeared blue against a clear background.Positive plaques, designated vT194, were picked from the cell monolayerand their progeny were replated. Five rounds of plaque isolation andreplating in the presence of Bluo-Gal resulted in the purification ofthe desired recombinant. The genomic structure of recombinant fowlpoxvT194 is depicted in FIG. 7B.

EXAMPLE 10 Generation of Recombinant Vaccinia, rV-TRICOM (hu) No. vT224

For the generation of rV-TRICOM(hu), a plasmid vector, designatedpT5064, was constructed to direct insertion of the foreign sequencesinto the thymidine kinase (TK) gene, which is located in the Hind III Jregion of the vaccinia genome. The human LFA-3 gene is under the controlof the 30K promoter, the human ICAM-1 gene is under the control of the13 promoter, and the human B7.1 gene is under the control of the sE/Lpromoter. In addition, the E. coli lacZ gene, under the control of theC1 promoter, is included as a screen for recombinant progeny. Theseforeign sequences are flanked by DNA sequences from the Hind III Jregion of the vaccinia genome (see FIG. 8). A plaque-purified isolatefrom the Wyeth (New York City Board of Health) strain of vaccinia wasused as the parental virus for this recombinant vaccine. The generationof recombinant vaccinia virus was accomplished via homologousrecombination between vaccinia sequences in the Wyeth vaccinia genomeand the corresponding sequences in pT5064 in vaccinia-infected CV-1cells (ATTC, CCL 70) transfected with pT5064. Recombinant virus wasidentified using the chromogenic assay for the lacZ gene productdescribed above. Viral plaques expressing lacZ appeared blue against aclear background. Positive plaques, designated vT224, were picked fromthe cell monolayer and their progeny were replated. Five rounds ofplaque isolation and replating in the presence of Bluo-Gal resulted inthe purification of the desired recombinant. The genomic structure ofrecombinant vT224 is depicted in FIG. 9A.

EXAMPLE 11 Generation of Recombinant Vaccinia rV-TAA/TRICOM(hu)

For the generation of rV-TAA/TRICOM(hu), a plasmid vector is constructedto direct insertion of the foreign sequences into the thymidine kinase(TK) gene, which is located in the Hind III J region of the vacciniagenome. The TAA gene, the human LFA-3 gene, the human ICAM-1 gene, thehuman B7.1 gene, and the E. coli lacZ gene are under the control of amultiplicity of poxvirus promoters. These foreign sequences are flankedby DNA sequences from the Hind III J region of the vaccinia genome. Aplaque-purified isolate from the Wyeth (New York City Board of Health)strain of vaccinia is used as the parental virus for this recombinantvaccine. The generation of recombinant vaccinia virus is accomplishedvia homologous recombination between vaccinia sequences in the Wyethvaccinia genome and the corresponding sequences in the plasmid vector invaccinia-infected CV-1 cells (ATTC, CCL 70) transfected with theplasmid. Recombinant virus is identified using the chromogenic assay forthe lacZ gene product described above. Viral plaques expressing lacZappear blue against a clear background. Positive plaques are picked fromthe cell monolayer and their progeny are replated. Additional rounds ofplaque isolation and replating in the presence of Bluo-Gal result in thepurification of the desired recombinant.

EXAMPLE 12 Generation of Recombinant Fowlpox rF-TAA/TRICOM(hu)

For the generation of rF-TAA/TRICOM(hu), a plasmid vector is constructedto direct insertion of the foreign sequences into the BamHI J region ofthe fowlpox genome. The TAA gene, the human LFA-3 gene, the human ICAM-1gene, the human B7.1 gene, and the E. coli lacZ gene are under thecontrol of a multiplicity of poxvirus promoters. These foreign sequencesare flanked by DNA sequences from the BamHI J region of the fowlpoxgenome. A plaque-purified isolate from the POXVAC-TC (Schering-PloughCorporation) strain of fowlpox is used as the parental virus for thisrecombinant vaccine. The generation of recombinant fowlpox virus isaccomplished via homologous recombination between fowlpox sequences inthe fowlpox genome and the corresponding sequences in the plasmid vectorin fowlpox-infected primary chick embryo dermal cells transfected withthe plasmid vector. Recombinant virus is identified using thechromogenic assay for the lacZ gene product described above. Viralplaques expressing lacZ appear blue against a clear background. Positiveplaques are picked from the cell monolayer and their progeny arereplated. Additional rounds of plaque isolation and replating in thepresence of Bluo-Gal result in the purification of the desiredrecombinant virus.

EXAMPLE 13 Generation of Recombinant Vaccinia Virus, rV-CEA(6D)/TRICOM(hu) No. vT238

For the generation of rV-CEA(6D)/TRICOM(hu), a plasmid vector,designated pT8016, was constructed to direct insertion of the foreignsequences into the thymidine kinase (TK) gene, which is located in theHind III J region of the vaccinia genome. The CEA gene was altered by invitro mutagenesis to express full-length protein containing one modifiedepitope. This mutation changed the encoded amino acid at position 576from asparagine to aspartic acid. The modified gene, designated CEA(6D),was designed to enhance the immunogenicity of CEA (Zaremba et al, 1997,Cancer Res. 57:4570-4577). The CEA(6D) gene is under the control of the40K promoter. The human LFA-3 gene is under the control of the 30Kpromoter, the human ICAM-1 gene is under the control of the I3 promoter,and the human B7.1 gene is under the control of the sE/L promoter. Inaddition, the E. coli lacZ gene, under the control of the C1 promoter,is included as a screen for recombinant progeny. These foreign sequencesare flanked by DNA sequences from the Hind III J region of the vacciniagenome (see FIG. 10). A plaque-purified isolate from the Wyeth (New YorkCity Board of Health) strain of vaccinia was used as the parental virusfor this recombinant vaccine. The generation of recombinant vacciniavirus was accomplished via homologous recombination between vacciniasequences in the Wyeth vaccinia genome and the corresponding sequencesin pT8016 in vaccinia-infected CV-1 cells (American Type CultureCollection (ATCC), Rockville, Md., CCL 70) transfected with pT8016.Recombinant virus was identified using the chromogenic assay for thelacZ gene product described above. Viral plaques expressing lacZappeared blue against a clear background. Positive plaques, designatedvT238, were picked from the cell monolayer and their progeny werereplated. Six rounds of plaque isolation and replating in the presenceof Bluo-Gal resulted in the purification of the desired recombinant. Thegenomic structure of recombinant vaccinia virus vT238 is shown in FIG.11.

EXAMPLE 14 Generation of Recombinant Fowlpox Virus, rF-TRICOM(mu) No.vT251

For the generation of rF-TRICOM(mu), a plasmid vector, designatedpT8019, was constructed to direct insertion of the foreign sequencesinto the BamHI J region of the fowlpox genome. The murine LFA-3 gene isunder the control of the 30K promoter, the murine ICAM-1 gene is underthe control of the 13 promoter, the murine B7.1 gene is under thecontrol of the sE/L promoter, and the lacZ gene is under the control ofthe C1 promoter. These foreign sequences are flanked by DNA sequencesfrom the BamHI J region of the fowlpox genome (see FIG. 12). Aplaque-purified isolate form the POXVAC-TC (Schering-Plough Corporation)strain of fowlpox was used as the parental virus for this recombinantvaccine. The generation of recombinant fowlpox virus was accomplishedvia homologous recombination between fowlpox sequences in the fowlpoxgenome and the corresponding sequences in pT8019 in fowlpox-infectedprimary chick embryo dermal cells transfected with pT8019. Recombinantvirus was identified using the chromogenic assay for the lacZ geneproduct described above. Viral plaques expressing lacZ appeared blueagainst a clear background. Positive plaques, designated vT251, werepicked from the cell monolayer and their progeny were replated. Threerounds of plaque isolation and replating in the presence of Bluo-Galresulted in the purification of the desired recombinant. The genomicstructure of recombinant vaccinia virus vT251 is shown in FIG. 13A.

EXAMPLE 15 Generation of Recombinant Fowlpox Virus, rF-TRICOM(hu) No.vT232

For the generation of rF-TRICOM(hu), a plasmid vector, designatedpT5072, was constructed to direct insertion of the foreign sequencesinto the BamHI J region of the fowlpox genome. The human LFA-3 gene isunder the control of the 30K promoter, the human ICAM-1 gene is underthe control of the 13 promoter, the human B7.1 gene is under the controlof the sE/L promoter, and the lacZ gene is under the control of the C1promoter. These foreign sequences are flanked by DNA sequences from theBamHI J region of the fowlpox genome (see FIG. 14). A plaque-purifiedisolate from the POXVAC-TC (Schering-Plough Corporation) strain offowlpox was used as the parental virus for this recombinant vaccine. Thegeneration of recombinant fowlpox virus was accomplished via homologousrecombination between fowlpox sequences in the fowlpox genome and thecorresponding sequences in pT5072 in fowlpox-infected primary chickembryo dermal cells transfected with pT5072. Recombinant virus wasidentified using the chromogenic assay for the lacZ gene productdescribed above. Viral plaques expressing lacZ appeared blue against aclear background. Positive plaques, designated vT232 were picked fromthe cell monolayer and their progeny were replated. Four rounds ofplaque isolation and replating in the presence of Bluo-Gal resulted inthe purification of the desired recombinant. The genomic structure ofrecombinant vaccinia virus vT232 is shown in FIG. 13B.

EXAMPLE 16 Generation of Recombinant Fowlpox Virus, rF-MUC-1/TRICOM(mu)No. vT250

For the generation of rF-MUC-1/TRICOM(mu), a plasmid vector, designatedpT8020, was constructed to direct insertion of the foreign sequencesinto the BamHI J region of the fowlpox genome. A truncated version ofthe MUC-1 gene was used, consisting of the signal sequence, ten copiesof the tandem repeat sequence, and the 3′ unique coding sequence. (SEQID NO:41). The nucleotide sequence of the tandem repeat region wasaltered to minimize homology between the repeats without changing theamino acid sequence. The gene was contained on an 1881 bp fragment whichincludes the truncated coding sequence, 6 nucleotides of the 5′untranslated region, and 186 nucleotides of the 3′ untranslated region(Gendler et al, 1990, J. Biol. Chem. 265:15286-15293). The murine LFA-3gene is under the control of the 30K promoter, the murine ICAM-1 gene isunder the control of the 13 promoter, the murine B7.1 gene is under thecontrol of the sE/L promoter, and the lacZ gene is under the control ofthe C1 promoter. These foreign sequences are flanked by DNA sequencesfrom the BamHI J region of the fowlpox genome (see FIG. 15). Aplaque-purified isolate from the POXVAC-TC (Schering-Plough Corporation)strain of fowlpox was used as the parental virus for this recombinantvaccine. The generation of recombinant fowlpox virus was accomplishedvia homologous recombination between fowlpox sequences in the fowlpoxgenome and the corresponding sequences in pT8020 in fowlpox-infectedprimary chick embryo dermal cells transfected with pT8020. Recombinantvirus was identified using the chromogenic assay for the lacZ geneproduct described above. Viral plaques expressing lacZ appeared blueagainst a clear background. Positive plaques, designated vT250, werepicked from the cell monolayer and their progeny were replated. Fourrounds of plaque isolation and replating in the presence of Bluo-Galresulted in the purification of the desired recombinant. The genomicstructure of recombinant vaccinia virus vT250 is shown in FIG. 16A.

EXAMPLE 17 Generation of Recombinant Fowlpox Virus, rF-MUC-1/TRICOM(hu)No. vT242

For the generation of rF-MUC-1/TRICOM(hu), a plasmid vector, designatedpT2186 was constructed to direct insertion of the foreign sequences intothe BamHI J region of the fowlpox genome. A truncated version of theMUC-1 gene was used, as described in Example 16 above. The MUC-1 gene isunder the control of the 40K promoter. The human LFA-3 gene is under thecontrol of the 30K promoter, the human ICAM-1 gene is under the controlof the 13 promoter, the human B7.1 gene is under the control of the sE/Lpromoter, and the lacZ gene is under the control of the C1 promoter.These foreign sequences are flanked by DNA sequences from the BamHI Jregion of the fowlpox genome (see FIG. 17). A plaque-purified isolatefrom the POXVAC-TC (Schering-Plough Corporation) strain of fowlpox wasused as the parental virus for this recombinant vaccine. The generationof recombinant fowlpox virus was accomplished via homologous recombinantbetween fowlpox sequences in the fowlpox genome and the correspondingsequences in pT2186 in fowlpox-infected primary chick embryo dermalcells transfected with pT2186. Recombinant virus was identified usingthe chromogenic assay for the lacZ gene product described above. Viralplaques expressing lacZ appeared blue against a clear background.Positive plaques, designated vT242, were picked from the cell monolayerand their progeny were replated. Four rounds of plaque isolation andreplating in the presence of Bluo-Gal resulted in the purification ofthe desired recombinant. The genomic structure of recombinant vacciniavirus vT242 is shown in FIG. 16B.

EXAMPLE 18 Generation of Recombinant Fowlpox Virus,rF-CEA(6D)/TRICOM(hu) No. vT236

For the generation of rF-CEA(6D)/TRICOM(hu), a plasmid vector,designated pT2187, was constructed to direct insertion of the foreignsequences into the BamHI J region of the fowlpox genome. The CEA(6D)gene is under the control of the 40K promoter. The human LFA-3 gene isunder the control of the 30K promoter, the human ICAM-1 gene is underthe control of the 13 promoter, the human B7.1 gene is under the controlof the sE/L promoter, and the lacZ gene is under the control of the C1promote. These foreign sequences are flanked by DNA sequences from theBamHI J region of the fowlpox genome (see FIG. 18). A plaque-purifiedisolate from the POXVAC-TC (Schering-Plough Corporation) strain offowlpox was used as the parental virus for this recombinant vaccine. Thegeneration of recombinant fowlpox virus was accomplished via homologousrecombination between fowlpox sequences in the fowlpox genome and thecorresponding sequences in pT2187 in fowlpox-infected primary chickembryo dermal cells transfected with pT2187. Recombinant virus wasidentified using the chromogenic assay for the lacZ gene productdescribed above. Viral plaques expressing lacZ appeared blue against aclear background. Positive plaques, designated vT236, were picked fromthe cell monolayer and their progeny were replated. Eight rounds ofplaque isolation and replating in the presence of Bluo-Gal resulted inthe purification of the desired recombinant. The genomic structure ofrecombinant vaccinia virus vT236 is shown in FIG. 16C.

EXAMPLE 19 Generation of Recombinant Fowlpox Virus,rF-PSA/PSMA/TRICOM(hu) No. vT257

For the generation of rF-PSA/PSMA/TRICOM(hu), a plasmid vector,designated pT5080, was constructed to direct insertion of the foreignsequences into the BamHI J region of the fowlpox genome. The geneencoding PSA was isolated by polymerase chain reaction amplification ofcDNA derived from RNA from the human LNCaP cell line (CRL 1740, AmericanType Culture Collection (ATCC), Rockville, Md.). The gene was containedon a 1346 bp fragment which includes the entire coding sequence for PSA,41 nucleotides of the 5′ untranslated region, and 552 nucleotides of the3′ untranslated region (Lundwall and Lilja, 1987, FEBS Lett.214:317-322). The gene encoding PSMA was isolated by polymerase chainreaction amplification of cDNA derived from RNA from the human LNCaPcell line. The gene was contained on a 2298 bp fragment which includesthe entire coding sequence for PSMA, 26 nucleotides of the 5′untranslated region, and 19 nucleotides of the 3′ untranslated region(Israeli et al, 1993 Cancer Res. 53:227-230). The PSA gene is under thecontrol of the 40K promoter and the PSMA gene is under the control ofthe 7.5K promoter. The human LFA-3 gene is under the control of the 30Kpromoter, the human ICAM-1 gene is under the control of the 13 promoter,the human B7.1 gene is under the control of the sE/L promoter, and thelacZ is under the control of the C1 promoter. These foreign sequencesare flanked by DNA sequences from the BamHI J region of the fowlpoxgenome (see FIG. 19). A plaque-purified isolate from the POXVAC-TC(Schering-Plough Corporation) strain of fowlpox was used as the parentalvirus for this recombinant vaccine. The generation of recombinantfowlpox virus was accomplished via homologous recombination betweenfowlpox sequences in the fowlpox genome and the corresponding sequencesin pT5080 in fowlpox-infected primary chick embryo dermal cellstransfected with pT5080. Recombinant virus was identified using thechromogenic assay for the lacZ gene product described above. Viralplaques expressing lacZ appeared blue against a clear background.Positive plaques, designated vT257, were picked from the cell monolayerand their progeny were replated. Five rounds of plaque isolation andreplating in the presence of Bluo-Gal resulted in the purification ofthe desired recombinant. The genomic structure of recombinant vacciniavirus vT257 is shown in FIG. 16D.

EXAMPLE 20 Generation of Recombinant MVA Virus, rMVA-TRICOM(mu) No.vT264

Modified Vaccinia Ankara (MVA) is an attenuated derivative of the Ankarastrain of vaccinia virus (Meyer et al, 1991, J. Gen. Virol.72:1031-1038). The seed stock from the MVA vaccine used as smallpoxvaccine in humans was obtained from Dr. Anton Mayr (Institute forMedical Microbiology, Munich). The seed stock was plaque-purified twotimes on primary chick embryo dermal cells.

For the generation of rMVA-TRICOM(mu), a plasmid vector, designatedpT5085, was constructed to direct insertion of the foreign sequencesinto the deletion III region of the MVA genome (Meyer et al, 1991, J.Gen. Virol. 72:1031-1038). The murine LFA-3 gene is under the control ofthe 30K promoter, the murine ICAM-1 gene is under the control of the 13promoter, the murine B7.1 gene is under the control of the sE/Lpromoter, and the lacZ gene is under the control of the C1 promoter.These foreign sequences are flanked by DNA sequences from the deletionIII region of the MVA genome (see FIG. 20). A plaque-purified isolatefrom the MVA vaccine seed stock was used as the parental virus for thisrecombinant vaccine. The generation of recombinant MVA was accomplishedvia homologous recombinant between MVA sequences in the MVA genome andthe corresponding sequences in pT5085 in MVA-infected primary chickembryo dermal cells transfected with pT5085. Recombinant virus wasidentified using the chromogenic assay for the lacZ gene productdescribed above. Viral plaques expressing lacZ appeared blue against aclear background. Positive plaques, designated vT264 were picked fromthe cell monolayer and their progeny were replated. Four rounds ofplaque isolation and replating in the presence of Bluo-Gal resulted inthe purification of the desired recombinant. The genomic structure ofrecombinant MVA vT264 is shown in FIG. 21A.

EXAMPLE 21 Generation of Recombinant MVA Virus, rMVA-PSA/PSMA/TRICOM(hu)No. vT260

For the generation of rMVA-PSA/PSMA/TRICOM(hu), a plasmid vector,designated pT5084, was constructed to direct insertion of the foreignsequences into the deletion III region of the MVA genome. The PSA geneis under the control of the 40K promoter and the PSMA gene is under thecontrol of the 7.5K promoter. The human LFA-3 gene is under the controlof the 30K promoter, the human ICAM-1 gene is under the control of the13 promoter, the human B7.1 gene is under the control of the sE/Lpromoter, and the lacZ gene is under the control of the C1 promoter.These foreign sequences are flanked by DNA sequences from the deletionIII region of the MVA genome (see FIG. 22). A plaque-purified isolatefrom the MVA vaccine seed stock was used as the parental virus for thisrecombinant vaccine. The generation of recombinant MVA was accomplishedvia homologous recombination between MVA sequences in the MVA genome andthe corresponding sequences in pT5084 in MVA-infected primary chickembryo dermal cells transfected with pT5084. Recombinant virus wasidentified using the chromogenic assay for the lacZ gene productdescribed above. Viral plaques expressing lacZ appeared blue against aclear background. Positive plaques, designated vT260, were picked fromthe cell monolayer and their progeny were replated. Four rounds ofplaque isolation and replating in the presence of Bluo-Gal resulted inthe purification of the desired recombinant. The genomic structure ofrecombinant MVA vT260 is shown in FIG. 21B.

EXAMPLE 22 Recombinant Poxviruses

The individual recombinant vaccinia viruses containing either the geneencoding murine costimulatory molecule B7-1 (designated rV-B7-1) or thegene encoding murine Intercellular adhesion molecule-1 (designatedrV-ICAM-1) have been described (10, 11). The recombinant vaccinia viruscontaining the gene for murine CD48 [designated rV-LFA-3; murine CD48 isthe homologue of human LFA-3 (CD58) (6)] was constructed in a similarfashion to rV-B7-1 and rV-ICAM-1, and has been described (12). In eachof these single recombinant vaccinia viruses, the gene encoding thecostimulatory molecule was put under the control of the vaccinia virusearly/late 40K promoter (15), and the transgene was inserted into theHind III M region of the genome of the Wyeth strain of vaccinia virus asdescribed (13). Recombinant fowlpox viruses were constructed by theinsertion of foreign sequences into the BamHI J region of the genome ofthe POXVAC-TC (Schering Corporation) strain of fowlpox virus asdescribed (14). In recombinant viruses containing a single foreign gene,the gene is under control of the vaccinia 40K promoter. rV-B7-1/ICAM-1is a recombinant vaccinia virus that contains the murine B7-1 gene undercontrol of the synthetic early/late (sE/L) promoter (16) and the murineICAM-1 gene under control of the 40K promoter. rV-B7-1/ICAM-1/LFA-3 is arecombinant vaccinia virus that contains the murine LFA-3 gene undercontrol of the vaccinia 30K (M2L) promoter (17), the murine ICAM-1 geneunder control of the vaccinia 13 promoter (18), and the murine B7-1 geneunder control of the synthetic early/late (sE/L) promoter.rF-CEA/B7-1/ICAM-1/LFA-3 is a recombinant fowlpox virus that containsthe human carcinoembryonic antigen (CEA) gene under control of the 40Kpromoter, the murine B7-1 gene under control of the sE/L promoter, themurine LFA-3 gene under control of the 13 promoter, and the murineICAM-1 gene under control of the vaccinia 7.5K promoter (19).Non-recombinant vaccinia virus was designated V-Wyeth, whilenon-recombinant fowlpox virus was designated WT-FP.

EXAMPLE 23 Expression of Recombinant Costimulatory Molecules

To confirm that each of the recombinant vectors could express theappropriate transgene(s), the murine adenocarcinoma cell line MC38 wasinfected with the various recombinant vaccinia or fowlpox constructs,and cell-surface expression of the transgene(s) was demonstrated by flowcytometry (FIG. 23). Uninfected cells (data not shown) and cellsinfected with wild-type vaccinia failed to express any of the threecostimulatory molecules. This observation was confirmed by PCR (data notshown). In contrast, cells infected with rV-B7-1 became stronglypositive for B7-1 protein; cells infected with rV-ICAM-1 became positivefor ICAM-1; and cells infected with rV-LFA-3 became positive for LFA-3protein. Similar analysis of a construct containing two costimulatorymolecules (rV-B7-1/CAM-1) showed expression of B7-1 (78% positive with amean fluorescent intensity (MFI) of 1012) and ICAM-1 (70% positive witha MFI of 690). Moreover, cells infected with the vaccinia multiple-geneconstruct rV-B7-1/ICAM-1/LFA-3 co-expressed all three costimulatorymolecules. To determine if the recombinant fowlpox viruses expressedtheir recombinant proteins, MC38 cells were infected with the fowlpoxconstructs in a similar manner (FIG. 23). Again, cells infected withwild-type fowlpox virus WT-FP failed to express any costimulatorymolecule. Cells infected with rF-B7-1 became positive for B7-1 protein,and cells infected with rF-ICAM-1 became positive for ICAM-1 protein. ArF-LFA-3 vector was not constructed. However, cells infected with thefowlpox multiple-gene construct rF-CEA/B7-1/ICAM-1/LFA-3 co-expressedall three costimulatory molecules.

Characterization of Recombinant Viruses: Fluorescent Analysis of ProteinSurface Expression

The MC38 murine colonic adenocarcinoma cell line has been described(20). Confluent MC38 cells were infected with vaccinia constructs(V-Wyeth, rV-B7-1, rV-ICAM-1, rV-LFA-3, rV-B7-1/ICAM-1/LFA-3) or fowlpoxconstructs (WT-FP, rF-B7-1, rF-ICAM-1, rF-CEA/B7-1/ICAM-1/LFA-3) at 5MOI (multiplicity of infection; PFU/cell) for 5 hours. CEA was used inone rF construct as a marker gene only. After infection, cells wereharvested and immunostained with FITC conjugated monoclonal antibodies(MAb) specific for murine CD80 (B7-1), CD54 (ICAM-1), or CD48 (LFA-3;PharMingen, San Diego, Calif.). Cell fluorescence was analyzed with aFACSCAN cytometer (Becton Dickinson, Mountain View, Calif.) with theLysis II software.

In Vitro Costimulation Analysis

Female C57BL/6 mice (6-8 weeks old) were obtained from Taconic Farms(Germantown, N.Y.). Naïve T cells were isolated from spleensmechanically dispersed through 70 m cell strainers (Falcon, BectonDickinson, Franklin Lakes, N.J.) to isolate single cell suspensions, anderythrocytes and dead cells were removed by centrifugation overFicoll-Hypaque gradients (density=1.119 g/ml) (Sigma, St. Louis, Mo.).Populations consisting of approximately 95% T cells were obtained bypassage of splenic mononuclear cells over two nylon wool columnssequentially (Robbins Scientific Corp., Sunnyvale, Calif.). For certainexperiments, T cells were further fractionated into CD4⁺ and CD8⁺populations by negative selection utilizing anti-CD4 or anti-CD8paramagnetic beads (MiniMACS, Miltenyi Biotec, Auburn, Calif.). T cellswere added at 10⁵/well in 96-well flat-bottomed plates (Costar,Cambridge, Mass.). Stimulator cells consisted of uninfected MC38 cellsor cells infected for 5 hours with 5 MOI of vaccinia constructs(V-Wyeth, rV-B7-1, rV-ICAM-1, rV-LFA-3, rV-B7-1/ICAM-1/LFA-3) or fowlpoxconstructs (WT-FP, rF-B7-1, rF-ICAM-1, rF-CEA/B7-1/ICAM-1/LFA-3) fixedwith 2% paraformaldehyde and added at 104/well. Cells in all wells werecultured in a total volume of 200 μl of complete media (CM) [RPMI 1640with fetal calf serum (10%), glutamine (2 mM), sodium pyruvate (1 mM),Hepes (7 mM), gentamicin (50 μg/ml), 2-mercaptoethanol (50 μM), andnon-essential amino acids (0.1 mM), (Biofluids, Rockville, Md.)] in thepresence of several dilutions (5 to 0.625 μg/ml for 2 days) ofConcanavalin-A (Con A, Sigma). Control wells received T cells,stimulator cells and media only. For indicated experiments, plate-boundanti-CD3 (1.5 μg/well-0.012 μg/well) was substituted for Con A. Cellswere labeled for the final 12-18 h of the incubation with 1 Ci/well³H-Thymidine (New England Nuclear, Wilmington, Del.) and harvested witha Tomtec cell harvester (Wallac Incorporated, Gaithersburg, Md.). Theincorporated radioactivity was measured by liquid scintillation counting(Wallac 1205 Betaplate, Wallac, Inc.) The results from triplicate wellswere averaged and are reported as mean CPM±SEM. For indicatedexperiments, the in vitro costimulation analysis was performed in thepresence of either a MAb specific for the expressed costimulatorymolecule or the matching isotype control antibody (Armenian hamster IgG,polyclonal). Antibodies used to block T-cell proliferation were Hamsteranti-murine CD80 (B7-1; clone 16-10A1), Hamster anti-murine CD54(ICAM-1; clone 3E2), or Hamster anti-murine CD48 (BCM-1; clone HM48-1),all from PharMingen. All antibodies were used at 25 μg/ml finalconcentration.

Determination of Costimulatory Molecule Capacity

T cells and stimulator cells were prepared as described above. Fixedstimulator cells expressing one or more costimulatory molecules wereadded to wells in various ratios in combination withV-Wyeth-infected/fixed stimulator cells to a total of 10⁴/well. T cells(10⁵/well) were then added, and cells were cultured in a total volume of2001 of CM in the presence of 2.5 μg/ml Con A for 2 days and labeled forthe final 12-18 h of the incubation with 1 μCi/well ³H-Thymidine. Theincorporated radioactivity was measured by liquid scintillation countingas before.

Cytokine Analysis

CD4⁺ and CD8⁺ T-cell populations were prepared as described above andadded at 2.5×10⁶/well in a 6-well plate (Costar). Stimulator cellpopulations were prepared as above and added at 2.5×10⁵/well. Cells werecultured in a total volume of 5 ml of CM in the presence of 2.5 μg/mlCon A for 24 hours. Supernatant fluids were collected and analyzed formurine IL-2, IFNγ, TNF-α, GM-CSF, and IL-4 by capture ELISA as describedpreviously (21). Sensitivity of detection was 30, 100, 20, 20, and 20μg/ml, respectively.

RNA populations from stimulated cells were also analyzed by multiprobeRNAse protection assay (mpRPA). Defined riboprobes for murine cytokineswere purchased from PharMingen. Assays were performed as describedpreviously (22). Protected probe-tagged duplexes were separated byelectrophoresis on 6% polyacrylamide gels. Dried gels were exposed toBiomax film (Kodak) at −70° C. for 24-72 hours. Radioactivity containedin the bands was quantified using a Storm system phosphoimager(Molecular Dynamics, Sunnyvale, Calif.). The net CPM for a given bandwas calculated by the following formula [cpm of cytokine gene minus cpmof background] and was expressed as a percent of the housekeeping genetranscript L32.

EXAMPLE 24 B7-1, ICAM-1, and LFA-3 Cooperate Synergistically to EnhanceT-cell Proliferation

The B7-1, ICAM-1, and LFA-3 molecules have been shown individually tocostimulate T-cell proliferation. However, because they may be expressedsimultaneously on APC, it has been difficult to examine relative rolesof individual costimulatory molecules during the induction of T-cellproliferation (2). To analyze the contribution of B7-1, ICAM-1 and/orLFA-3 molecules to the induction of naïve T-cell proliferation, amodified in vitro model (23, 24) was employed where the first signal forT-cell activation was delivered via a pharmacological reagent (Con A). Apanel of stimulator cells that differed only in costimulatory moleculeswas created using the MC38 cell line infected with various recombinantvaccinia (FIG. 24A) or fowlpox (FIG. 24B) viruses engineered to expresscostimulatory molecules. The second, or “costimulatory,” signal wasdelivered to the T cell via one or more costimulatory moleculesexpressed on the surface of these “stimulator” MC38 cells. As shown inFIG. 24A, both uninfected MC38 cells and MC38/V-Wyeth induced marginalproliferation of T cells at all levels of Con A examined. MC38/LFA-3induced a small (2.1-fold) but significant (P<0.05) increase in T-cellproliferation. Delivery of signal-2 via MC38/ICAM-1 induced a 3.5-foldincrease in T-cell proliferation at 2.5 μg/ml Con A. MC38/B7-1 induced a7.8-fold and a 16-fold increase in proliferation at 2.5 and 1.25 μg/mlCon A respectively. However, MC38/B7-1/ICAM-1/LFA-3 (MC38 cellsco-expressing all three costimulatory molecules) induced a 17.5-foldincrease in T-cell proliferation at 2.5 μg/ml Con A, and a 34-foldincrease at 1.25 μg/ml Con A. Moreover, at low Con A levels (0.625μg/ml), expression of ICAM-1 and LFA-3 did not induce T-cellproliferation. While B7-1 induced measurable proliferation (20,000 CPM)at 0.625 μg/ml Con A, the co-expression of all three costimulatorymolecules induced an even greater level of proliferation (100,000 CPM)(FIG. 24A). These experiments were repeated four times with similarresults.

MC38 stimulator cells were also prepared by infection with recombinantfowlpox vectors (FIG. 24B). Again, uninfected MC38 or MC38/WT-FP inducedmarginal proliferation of T cells at all levels of Con A examined.MC38/rF-ICAM-1 supported a 2-fold increase, MC38/rF-B7-1 supported a3.2-fold increase, and MC38/rF-B7-1/ICAM-1/LFA-3 supported a 6-foldincrease in T-cell proliferation at 2.5 μg/ml Con A. Similar resultswere obtained when this experiment was repeated two additional times.Similar results were also observed when the first signal was deliveredvia immobilized anti-CD3 (data not shown). The differences noted inproliferation supported by MC38/rV-B7-1/ICAM-1/LFA-3 andMC38/rF-CEA/B7-1/ICAM-1/LFA-3 (17.5-fold vs. 6-fold) are most likely dueto the levels of expressed recombinant protein(s) following a 5-hourinfection period (FIG. 23). Specifically, approximately 70% of the cellsinfected with rV-B7-1/ICAM-1/LFA-3 express the costimulatory molecules,while approximately 40% of cells infected with rF-CEA/B7-1/ICAM-1/LFA-3are positive. Those positive cells infected with the rF vectors expressrecombinant B7-1 and ICAM-1 at levels of 50% of those cells infectedwith rV-B7-1/ICAM-1/LFA-3 with the conditions used.

EXAMPLE 25 Specificity of Costimulatory Molecule Contribution on T-CellProliferation

To further confirm the specificity of the proliferative contribution ofB7-1, ICAM-1, or LFA-3, MC38 stimulator cells were again prepared byinfection with V-Wyeth, rV-B7-1, rV-ICAM-1, or rV-LFA-3 and co-culturedwith naive murine T cells and Con A in the presence or absence of MAbspecific for the given costimulatory molecule. As shown in FIG. 3B,MC38/B7-1 enhanced T-cell proliferation 4.5-fold more than that ofMC38/V-Wyeth (FIG. 25A). This increased proliferation was inhibited 83%by the addition of a blocking MAb for murine B7-1. Similarly,MC38/ICAM-1 (FIG. 25C) increased proliferation 2.25-fold, which was thenreduced by 88% in the presence of anti-murine ICAM-1 MAb. Finally,MC38/LFA-3 (FIG. 25D) increased proliferation 2.1-fold, which was thenreduced by 98% in the presence of anti-murine CD48 MAb. For each group,incubation with the appropriate isotype control antibody (as specifiedin Materials and Methods) failed to block the noted proliferation. Thisexperiment was repeated two additional times with similar results.

EXAMPLE 26 Determination of Costimulatory Molecule Capacity

Modification of the in vitro costimulation assay allowed a quantitativeestimation of the relative capacity of B7-1, ICAM-1, and/or LFA-3 todeliver the second signal for T-cell proliferation. To that end,stimulator cells (MC38 cells infected with the various recombinantvaccinia viruses) were titered out by dilution with varying amounts ofMC38 cells infected with V-Wyeth and co-cultured with a constant numberof T cells in the presence of 2.5 μg/ml Con A. The MC38 to T-cell ratioin these experiments remained constant at 1:10. As seen in FIG. 4,MC38/LFA-3 (closed triangles) enhanced proliferation of T cells overthat of MC38/V-Wyeth (open square) out to a concentration of 40% (i.e.,of the stimulator cells in the well, 40% were infected with rV-LFA-3 andthe remaining 60% were infected with V-Wyeth). MC38/ICAM-1 (closedcircles) or MC38/B7-1 (closed diamonds) supported increasedproliferation out to a concentration of 13% and 6%, respectively. Incontrast, MC38/B7-1/ICAM-1/LFA-3 enhanced proliferation when less than3% of stimulator cells contained the triad vector (extrapolated to lessthan 1% via linear least squares analysis). Given the titration curvesof these individual costimulatory molecules, it appeared that the extentof T-cell proliferation mediated by ICAM-1 and B7-1 is 3-fold and6-fold, respectively, more potent than that mediated by LFA-3 alone.Clearly the strongest proliferation, however, is mediated byB7-1/ICAM-1/LFA-3. It should be noted (FIG. 26) that at relatively lowstimulator cell concentrations (i.e., when 3%-6% of the MC38 cells areacting as stimulator cells), expression of LFA-3, ICAM-1, and even B7-1alone does not enhance T-cell activation, while the three costimulatorymolecules expressing stimulator cells substantially enhance T-cellactivation. The data in FIG. 26 (insert) shows proliferation resultsobtained when 3% of the MC38 stimulator cells were infected with thevectors denoted. Since each well contained 10⁴ total MC38 cells and 10⁵naive T cells, the actual stimulator to T-cell ratio in these cultureswas 0.003. Note that the MC38 cells infected with the two-gene construct(rV-B7-1/ICAM-1) induced little, if any, proliferation of T cells underthese conditions, while MC38/B7-1/ICAM-1/LFA-3 increased proliferationsubstantially (p<0.0001).

EXAMPLE 27 Costimulation of CD4⁺ and CD8⁺ T Cells

To further characterize the T-cell response to costimulatory moleculesexpressed singly or in combination, the ability of B7-1, ICAM-1, andLFA-3 to costimulate purified CD4⁺ and CD8⁺ T cells was tested. FIG. 5shows the proliferation of purified CD4⁺ (FIG. 27A) and CD8⁺ (FIG. 27B)cells activated with suboptimal concentrations of Con A. Thestratification of stimulator cell effects on proliferation was similarfor both CD4⁻ and CD8⁺ cells: MC38/LFA-3 stimulated the weakestproliferation, followed by MC38/ICAM-1 and MC38/B7-1.MC38/B7-1/ICAM-1/LFA-3 were the most potent stimulator cells for CD4⁺and CD8⁺ T cells. These experiments were repeated three additional timeswith similar results. It should be noted that at very low concentrationsof Con A (0.625 μg/ml, FIG. 5, panels C and D), there was no significantenhancement in activation of CD4⁺ or CD8⁺ T cells when either ICAM-1,LFA-3, B7-1, or the B7-1/ICAM-1 combination was used to provide thesecond signal. However, substantial activation of both T-cell subsetswas observed when the vaccinia virus coexpressing the triad ofcostimulatory molecules was employed. Similar results were noted whenthe first signal was delivered via immobilized anti-CD3 (data notshown).

It has been reported that B7-1 costimulation prolongs IL-2 mRNA halflife and upregulation of EL-2 transcription, resulting in production ofconsiderable amounts of secreted IL-2 (4, 25). Additionally, T-cellcostimulation with LFA-3 has been reported to have an effect on avariety of cytokines, notably IL-2 and IFN-γ (6). To determinequalitative and quantitative effects of costimulation by single ormultiple costimulatory molecules on cytokine production, purified CD4⁺and CD8⁺ T cells were again co-cultured with various stimulator cellsexpressing B7-1, ICAM-1, and LFA-3 alone or in combination in thepresence of 2.5 μg/ml Con A. Supernatant fluids were analyzed for IL-2,IFN-γ, TNF-α, GM-CSF, and IL-4 after 24 hours. Uninfected MC38 (data notshown) and MC38N-Wyeth induced a marginal quantity of IL-2 from CD4⁺cells (FIG. 28A), while MC38/B7-1 induced 3,979 pg/ml. However, T-cellstimulation with MC38/B7-1/ICAM-1/LFA-3 induced a 10-fold greater amountof IL-2. Similarly, MC38/B7-1 induced a marginal quantity of IL-2 fromCD8⁺ cells (FIG. 28B), while MC38/B7-1/ICAM-1/LFA-3 induced a 20-foldgreater amount (6,182 pg/ml). IFN-γ production by stimulated T cells wasalso examined. MC38/B7-1 and MC38/LFA-3 induced only moderate amounts ofIFN-γ from CD4′ cells (FIG. 28C). In contrast, stimulation of CD4⁺ cellswith MC38/B7-1/ICAM-1/LFA-3 induced 4-fold more IFN-γ than stimulationwith any other construct. Stimulation of CD8⁺ cells withMC38/B7-1/ICAM-1/LFA-3 induced the greatest amount of IFN-γ greater than6-fold more than CD8⁺ cells stimulated with any of the other constructs(FIG. 28D). Stimulation of either cell type with any construct failed tomediate significant changes (p>0.05) in the levels of secreted TNF-αGM-CSF, or IL-4 (data not shown). It appears that the predominantculmination of stimulation via the triad construct(rV-B7-1/ICAM-1/LFA-3) was IL-2 secretion from CD4⁺ cells and IFN-γsecretion from CD8⁺ T cells. These experiments were repeated threeadditional times with similar results. Studies were also carried outcomparing stimulator cells infected with the two-gene construct(rV-B7-1/ICAM-1) vs. the multi-gene construct (rV-B7-1/ICAM-1/LFA-3) fortheir ability to enhance cytokine production by T cells. Only smalldifferences were observed between the two in IFN-γ production by eitherCD4⁺ or CD8⁺ cells, or in IL-2 production by CD8⁺ cells. But asubstantial difference was seen in the stimulation of IL-2 production byCD4⁺ cells (5000 pg/ml employing MC38/B7-1/ICAM-1 vs. 39,600 pg/mlemploying MC38/B7-1/ICAM-1/LFA-3).

Cytokine expression from CD4⁺ and CD8⁺ T cells stimulated with single ormultiple costimulatory molecules was also analyzed at the RNA levelutilizing the multiprobe RNAse protection assay (mRPA). A representativeradiographic profile and quantitative analysis from two independentexperiments are depicted (FIG. 29). Levels of IL-4, IL-5, IL-10, IL-15,and IL-6 were similar in CD4′ T cells stimulated with MC38/V-Wyeth,MC38/B7-1, MC38/ICAM-1, MC38/LFA-3, or MC38/B7-1/ICAM-1/LFA-3 (FIG. 29,panel B histogram). IL-2 and IFN-γ expression levels were highest inCD4⁺T cells stimulated with MC38/B7-1/ICAM-1/LFA-3 when compared withCD4′ cells stimulated with MC38 cells expressing any singlecostimulatory molecule (FIG. 29B). Slightly higher levels of IL-13,IL-9, and IL-6 were also noted in CD4 cells stimulated withMC38/B7-1/CAM-1/LFA-3. Expression of cytokine genes was also analyzed instimulated CD8⁺T cells. Of the cytokine RNAs analyzed, IL-2 andparticularly IFN-γ levels were significantly higher when these cellswere stimulated with MC38/B7-1/ICAM-1/LFA-3, compared to T cellsstimulated with MC38 cells expressing any single costimulatory molecule.Thus, the predominant synergistic effect of the triad of costimulatorymolecules in cytokine production was IL-2 in CD4⁺ cells and IFN-γ inCD8⁺ T cells.

EXAMPLE 28 Effect of TRICOM Costimulation on Apoptosis of Stimulated Tcells Apoptosis Studies

To determine if stimulation of T cells with signal 1 and rV-TRICOM wouldlead to cell survival or programmed cell death (PCD), CD8⁺ T cells wereactivated with Con A for signal 1, cultured with either V-WT, rV-B7-1 orrV-TRICOM-infected MC38 cells for 48 hr, and replated for 24 hr inmedium to measure apoptosis. Apoptosis was assessed using the TUNELassay, as described by Gavrieli, Y et al. J. Cell Biol 119: 493-501,1992. T cells activated by the combination of MC38 and Con A orMC38/V-WT and Con A in the absence of costimulatory signals exhibitedhigh levels of spontaneous apoptosis (82.9±1, respectively). T cellsactivated by Con A and MC38/B7-1 or Con A and MC38/TRICOM exhibitedsubstantially less spontaneous apoptosis (31.3±3.8 and 30.7±1,respectively).

The results clearly demonstrate apoptosis in T cells stimulated withMC38 cells in the presence of Con A with or without V-WT infection(i.e., in the absence of signal 2). While Con A with MC38/TRICOM clearlystimulated CD8′ cells to far greater levels than Con A with MC38/B7-1and resulted in the production of higher levels of IFN-γ and IL-2, thisdid not result in any greater degree of apoptosis.

EXAMPLE 29 Anti-tumor Effect of rV-CEA/TRICOM In Vivo

Studies were conducted to determine if an antigen-specific immuneresponse could be enhanced using a TRICOM vector. A four-gene vacciniarecombinant was constructed that contained the human CEA gene and theB7-1, ICAM-1 and LFA-3 genes, designated rV-CEA/TRICOM, as disclosedherein. Six to eight-week-old female C57 BL/6 mice (Taconic Farms) orC57BL/6 mice transgenic for human CEA (Kass, E et al Cancer Res. 59:676-683, 1999) were vaccinated by tail scarification with either Hank'sBalanced Salt Solution (HBSS) or one time with 10⁷ pfu rV-CEA,rV-CEA/B7-1 or rV-CEA/TRICOM, and spleens were harvested 22 days later.Lymphoproliferative activity of splenocytes was analyzed as describedpreviously (5).

As seen in FIG. 30 (insert), splenic T cells of mice vaccinated withrV-TRICOM showed higher levels of CEA-specific stimulation compared withT cells obtained from mice vaccinated with rV-CEA; Ovalbumin and Con Awere used as controls. An experiment was then conducted to determine ifrV-CEA/TRICOM could induce long-term immunity. Mice (5/group) werevaccinated one time with either V-WT, rV-CEA, or rV-CEA/TRICOM. Onehundred days later, mice were challenged with a high dose (1×10⁶) ofMC38 colon carcinoma cells expressing CEA (5). All mice receiving V-WTand rV-CEA succumbed to tumors, while all mice vaccinated with rV-TRICOMwere alive 50 days post-challenge (FIG. 30).

CEA-transgenic mice (Kass 1999, ibid; Thompson, J. A. et al. J. Clin.Lab. Anal. 5:344-366, 1999) in which the human CEA gene is expressed innormal adult gastrointestinal tissue, and whose serum is CEA-positive,were employed to determine if the rV-CEA/TRICOM vector could enhanceT-cell responses to a self-antigen. CEA transgenic mice were separatedinto 5 mice/group. Two mice were vaccinated once with 10⁷ pfu rV-CEA,rV-CEA/B7-1, rV-CEA/TRICOM or buffer and were euthanized on day 30 toanalyze CEA-specific T-cell responses. T-cell responses obtained aftervaccination with rV-CEA/TRICOM were substantially greater than thoseobtained with rV-CEA (Table 2). Responses to ovalbumin and Con A wereused as controls. The remaining 3 CEA-transgenic mice in each group wereused to determine if anti-tumor responses to a CEA-expressing tumorcould be enhanced employing a TRICOM vector. These mice were firstinoculated s.c. with 4×10⁵ MC38 carcinoma cells expressing the CEA gene(5). Four days later, mice were vaccinated one time at a distal sitewith 10 pfu viral recombinant or buffer. No tumors grew in micevaccinated with rV-CEA/TRICOM, whereas tumors continued to grow in micevaccinated with buffer, rV-CEA and rV-CEA/B7-1 (Table 2). These resultssupport the in vivo activity of TRICOM vectors.

TABLE 2 Enhanced Immune Response and Anti-Tumor Response ofrV-CEA/TRICOM in CEA Transgenic Mice Stimulation Index (SI) Con A OvalCEA CEA Tumor Value Immunogen (5 μg/ml) (100 μg/ml) (100 μg/ml) (25μg/ml) Day 14 Day 35 HBSS 109 1.0 1.3 2.0 698 ± 928 3,674 ± 3,107 rV-CEA123 0.9 4.9 4.0 259 ± 0  1,112 ± 1,685 rV-CEA/B7-1 93 1.3 7.1 4.3 150 ±236 2,696 ± 1,936 rV-CEA/TRICOM 111 1.1 19.2 15.9 0 ± 0 ±0 C57BL/6CEA-transgenic mice (5 per group) were vaccinated via skin scarificationwith buffer or vaccinia recombinant (10⁷ pfu) one time on Day 0. On Day30, 2 mice were killed and splenic T cells were analyzed for T-cellproliferative responses. Each value represents the SI of the mean CPM oftriplicate samples versus media. Standard deviation never exceeded 10%.On Day −4, 3 mice per group were given 4 × 10⁵ MC38 colon carcinomacells expressing CEA. Tumor volume is given at Days 14 and 35post-vaccination.

EXAMPLE 30 Costimulation of CD4⁺ and CD8⁺ T cells by ProgenitorDendritic Cells and Dendritic Cells Infected with rV-B7/ICAM-1/LFA-3

Fresh CD34⁺ bone marrow cells (dendritic cell precursors) were obtainedfrom C57BL/6 mice by the method of Inaba et al (41). These precursorcells were either used immediately or cultured for 6 days in GM-CSF andIL-4 (42) to generate mature dendritic cells (DC). CD34⁺ precursor cellsand DC were infected for 18 hours with the recombinant vaccinia virusencoding multiple costimulatory molecules rV-B7/ICAM-1/LFA-3(rV-Tricom), 10 MOI. After 5 hours of infection, a sample of cells wereharvested and a phenotypic analysis was performed. Dendritic cells arethough of in the art as the ‘ultimate’ APC, expressing a large array ofcostimulatory molecules at high levels. Table 3 shows that murine DCindeed express the costimulatory molecules B7-1, B7-2, ICAM-1, and LFA-3at relatively high levels (mean fluorescent intensity, MFI; depicted inparenthesis). However, when DC were infected with rV-B7/ICAM-1/LFA-3,there was a significant increase in both the level of costimulatorymolecule expression as well as the percentage of cell expressing themultiple costimulatory molecules. The percentage of cells expressingB7-1 increased from 65% to 86%, while the MFI increased 4-fold; thepercentage of cells expressing ICAM-1 increased from 32% to 68%, whilethe MFI increased 2.5 fold; the percentage of cells expressing LFA-3increased from 44% to 75%.

TABLE 3 Phenotypic Analysis of Progenitor DC Pre and Post Infection¹with rV-COS² Marker Infection H2-K^(b) I-A^(b) CD11b CD11c B7-2 B7-1ICAM-1 LFA-3 None   90³  64  63  29  38  65  32  44  (994)⁴ (621) (397)(223) (319) (300) (336) (378) V-Wyeth  75  60  59  27  36  65  33  43(554) (633) (398) (218)  317) (311) (296) (322) rV-B7  76  67  70  34 41  83  43  51 (516) (755) (419) (213) (320) (661) (363) (333)rV-B7/ICAM/  79  63  63  30  42  86  68  75 LFA-3 (579) (696) (408)(203) (360) (1253)  (810) (484) ¹5 hour infection at 10 MOI ²rV-COS =recombinant vaccinia encoding a foreign costimulatory molecule. ³= %cells expressing marker ⁴= mean fluorescent intensity

For use as stimulator cells, the infected CD34′ precursor cells and DCwere irradiated (2000 rad) and used to stimulate naïve CD4⁺ and CD8⁺T-cells in the presence of Con A as outlined in FIG. 31.

Progenitor dendritic cells infected with recombinant poxvirus encodingB7.1, ICAM-1, and LFA-3 were able to stimulate both CD4⁺ and CD8⁺ Tcells. The stimulation of CD8⁺ T cells by the B7.1, ICAM-1, LFA-3expressing progenitor dendritic cells was greater than that achievedusing non-infected mature CD34⁺ dendritic cell (FIG. 32). Moreover,infection and expression of the three costimulatory molecules in matureCD34⁺ dendritic cells (pretreated with IL-4 and GM-CSF) resulted in adramatic increase in stimulation of both CD4⁺ and CD8⁺ T cells (FIG.33).

One skilled in the art can also measure the quality of a dendritic cellpopulation by its ability to support an alloreactive response (mixedlymphocyte reaction, MLR) (43). FIG. 34 shows the results of a mixedlymphocyte culture using dendritic cells infected with rV-TRICOM. Themixed lymphocyte reaction uses DCs from C57BL/6 mice which arestimulating T lymphocytes from Balb/c, (i.e. an anti-allotype reaction).

These data show that the degree of proliferation in a mixed lymphocytereaction is dramatically higher using DCs infected with rV-TRICOM ascompared to uninfected DCs or DCs infected with wild-type vaccinia.

FIG. 35 demonstrates that DCs infected with rV-TRICOM are far superiorthan standard DCs in stimulating a CEA peptide-specific murine T cellline. This T-cell line is CD8⁺ and is specific for the CEA D^(b) Class-Irestricted epitope EAQNTTYL (CAP-M8). The combination of DCs pulsed withthe CEA peptide (1 μg/ml) and previously infected with rV-TRICOM isclearly superior in stimulating CEA-specific T cell responses,especially at low T-cell to DC ratios.

EXAMPLE 31 Murine T Cell Stimulation In Vitro and In Vivo Using rV- orrF-TRICOM Infected Murine Bone Marrow-Derived Dendritic CellsExperimental Protocol Peptides

The H-2k^(b)-restricted peptides OVA (ovalbumin₂₅₇₋₂₆₄, SIINFEKL)⁴¹ andVSVN (vesicular stomatitis virus N₅₂₋₅₉, RGYVYQGL)⁴², and the H-2 D^(b)restricted peptides CAP-M8 (CEA₅₂₆₋₅₃₃), EAQNTTYL) and FLU-NP(NP₃₆₆₋₃₇₄,ASNENMDAM)⁴³ were either purchased (Multiple Peptide Systems, San Diego,Calif.) or synthesized in-house (Applied Biosystems 432A Synergy PeptideSynthesizer, Foster City, Calif.).

Cell Lines and Cell Cultures

The OVA and Cap-M8 CD8⁺ cytotoxic T-cell lines were generated in-housefrom C57BL/6 mice and recognize the OVA and Cap-M8 peptides,respectively. The CTL lines were maintained by weekly in vitrostimulation cycles with irradiated naïve splenocytes in complete medium(CM) [RPMI 1640 with fetal calf serum (10%); glutamine (2 mM), sodiumpyruvate (1 mM), Hepes (7 mM), gentamicin (50 μg/ml), 2-mercaptoethanol(50 μM), and non-essential amino acids (0.1 mM), (Biofluids, Rockville,Md.)], supplemented with 1 μg/ml specific peptide and 10 U/ml murineIL-2 (Boehringer Mannheim, Indianapolis, Ind.). Twenty-four hours priorto using these cells as responders in antigen-specific proliferationassays, the cells were purified by centrifugation over a Ficoll-Hypaquegradient (density=1.119 g/ml, Sigma Chemical Co., St. Louis, Mo.) andreplated in six-well culture plates (10⁶ cells/ml, 5 ml/well) in CMsupplemented with 10 U/ml murine IL-2 only. For cytotoxicity assays, thetarget tumor-cell line used was EL4 (C57BL/6, H-2b, thymoma, ATCCTIB-39).

DC Preparation

Bone marrow, was derived from six- to eight-week-old female C57BL/6 mice(Taconic Farms, Germantown, N.Y.). The procedure used in this study wasa slightly modified version of that described by Inaba et al.⁴¹.Briefly, bone marrow was flushed from the long bones of the limbs andpassed over a Ficoll-Hypaque gradient. Bone-marrow cells were depletedof lymphocytes and 1a⁺ cells using a cocktail of magnetic beads specificfor CD4, CD8, and anti-MHC Class-II (MiniMACS, Miltenyi Biotec, Auburn,Calif.). Cells were plated in six-well culture plates (10⁶ cells/ml, 5ml/well) in CM supplemented with 10 ng/ml GM-CSF and 10 ng/ml IL-4 (R&DSystems, Minneapolis, Minn.). Cells were replated in freshcytokine-supplemented media on days 2 and 4. At 6 days of culture, cellswere harvested for infection, analysis and immunizations. For specifiedexperiments, DC were treated with murine TNF-α (100 ng/ml, BoehringerMannheim, Indianapolis, Ind.) or CD40 mAb (5 μg/ml, PharMingen, SanDiego, Calif.) during the final 24 h of culture.

Recombinant Poxviruses

The rV virus containing the gene that encodes the murine costimulatorymolecule B7-1 (CD80) under control of the synthetic early/late (sE/L)promoter (designated rV-B7-1) has been described herein. The rV viruscontaining the murine LFA-3 gene (CD48) under control of the vaccinia30K (M2L) promoter, the murine ICAM-1 (CD54) gene under control of thevaccinia I3 promoter, and the murine B7-1 gene under control of thesynthetic early/late (sE/L) promoter has been designated rV-TRICOM. Thevectors rF-B7-1 and rF-B7-1/ICAM-1/LFA-3 (designated rF-TRICOM) are rFviruses that were constructed similarly to rV-B7-1 and rV-TRICOM,respectively. A fowlpox-TRICOM construct containing a reporter gene,human CEA, was used in certain experiments. Non-recombinant wild-typevaccinia virus (Wyeth strain) was designated V-WT, while wild-typefowlpox virus was designated FP-WT.

Infection of DC

DC were harvested on day 6 and washed with Opti-Mem (Gibco-BRL,Gaithersburg, Md.). The cells were then either mock-infected with HBSS;infected with V-WT, rV-B7, or rV-TRICOM at 25 MOI (multiplicity ofinfection; PFU/cell); or infected with FP-WT, rF-B7-1, or rF-TRICOM at50 MOI in Opti-Mem for 5 h. Warn CM was added after infection, and thecells were incubated at 37° C. overnight. After infection, the cellswere harvested for immunostaining, in vitro costimulation analysis, andin vivo administration.

Flow Cytometric Analysis

Cell-surface staining utilized three-color immunofluorescence. Stainingwas performed with primary FITC-labeled antibodies CD11c, CD11b, H-2K^(b), H-2D^(b), CD19, Pan-NK; primary PE-labeled antibodies IA^(b),CD48 (mLFA-3), CD86 (B7-2), CD3, CD14; and the biotin-labeled antibodiesCD80 (B7-1), CD57 (ICAM-1), CD40. Biotin-labeled antibodies weresubsequently labeled with Cychrome-streptavidin. All antibodies werepurchased from PharMingen. Cell fluorescence was analyzed and comparedwith the appropriate isotype matched controls (PharMingen) with aFACSCAN cytometer (Becton Dickinson, Mountain View, Calif.) using theLysis II software.

In Vitro Costimulation Analysis: Pharmacological Signal-1

Female, six- to eight-week-old C57BL/6 mice were obtained (TaconicFamms, Germantown, N.Y.), and naive T cells were isolated as previouslydescribed⁵. T cells were added at 10⁵/well in 96-well, flat-bottomedplates (Costar, Cambridge, Mass.). Stimulator cells consisted of eitheruninfected DC, mock-infected DC, or DC infected with vaccinia vectors(V-WT, rV-B7-1, rV-TRICOM) or fowlpox vectors (FP-WT, rF-B7-1 orrF-TRICOM) irradiated (20 Gy) and added at 10⁴/well. Cells in all wellswere cultured in a total volume of 200 μl of CM in the presence ofseveral concentrations (2.5 to 0.9 μg/ml) of Con A (Sigma) for 2 days.Cells were labeled for the final 12-18 hr of the incubation with 1μCi/well ³H-Thymidine (New England Nuclear, Wilmington, Del.) andharvested with a Tomtec cell harvester (Wallac Incorporated,Gaithersburg, Md.). The incorporated radioactivity was measured byliquid scintillation counting (Wallac 1205 Betaplate, Wallac, Inc.). Theresults from triplicate wells were averaged and are reported as meanCPM±SEM.

Mixed-Lymphocyte Reaction

MLR was used to assess the stimulatory function of DC for allogeneic andsyngeneic naïve T cells. T cells were isolated from Balb/C or C57BL/6mice as before. Stimulator cells consisted of DC that were eitheruninfected; mock infected; or infected with V-WT, rV-137-1, rV-TRICOM,FP-WT, rF-B7-1 or rF-TRICOM and irradiated (20 Gy). T cells (5×10⁴/well)were co-cultured with graded numbers of stimulator cells in CM inflat-bottom 96-well culture plates and incubated at 37° C., 5% CO₂ for 4days, labeled for the final 12-18 hr of the incubation with 1 μCi/well³H-Thymidine, harvested, and analyzed as before.

In vitro Costimulation Analysis: Peptide-Specific Signal

Rested OVA or CAP-M8 T cells (responders) were added at 5×10⁴/well in96-well, flat-bottomed plates. Stimulator cells consisted of DC thatwere either uninfected, or infected with V-WT, rV-137-1, or rV-TRICOMand irradiated (20 Gy). Cells in all wells were cultured in a totalvolume of 200 μl of CM. The costimulation assay was carried out usingtwo sets of conditions: (1) a 10:1 fixed ratio of responder:stimulatorcells that were cultured in the presence of several concentrations ofspecific peptide or appropriate control peptide or (2) a fixedconcentration of specific peptide or control peptide cultured at variousresponder:stimulator cell ratios. Cells were cultured for 72 h, labeledfor the final 12-18 h of incubation with 1 μCi/well ³H-Thymidine,harvested, and analyzed as before.

CTL Induction In Vivo and Cytotoxic Analysis

DC (1×10⁶) that were either uninfected or infected with V-WT orrV-TRICOM were washed twice in Opti-Mem and resuspended in 1 ml of thesame medium containing 10 μM of either OVA or CAP-M8 peptides. After 2 hincubation at 37° C., cells were washed twice in HBSS and resuspended inHBSS for injections. Peptide-pulsed DC (1×10⁵ cell/mouse) were injected1-3 times intravenously at 7-day intervals. Control mice were immunizedsubcutaneously with 100 μg indicated peptide in Ribi/Detox adjuvant(Ribi ImmunoChem Research, Hamilton, Mont.). Fourteen days following thefinal inoculation, spleens from two animals per group were removed,dispersed into single-cell suspensions, pooled, and co-incubated with 10μg/ml of appropriate peptide for six days. Bulk lymphocytes wererecovered by centrifugation through a density gradient (LSM, OrganonTeknika, West Chester, Pa.). EL-4 cells were prepared for use as targetsin a standard cytolytic assay using ¹¹¹In, as previously⁴⁵. Target cellswere pulsed with 10 μM specific peptide for 1 hour at 37° C., while asecond group of target cells was pulsed with control peptide.Lymphocytes and peptide-pulsed targets (5×10³ cells/well) were suspendedin CM, combined at effector:target ratios of 80:1 to 10:1 in 96-wellU-bottomed plates (Costar) and incubated for 5 h at 37° C. with 5% CO₂.After incubation, supernatants were collected using a SupernatantCollection System (Skantron, Sterling, Va.), and radioactivity wasquantified using a gamma counter (Cobra Autogamma, Packard, DownersGrove, Ill.). The percentage of specific release of ¹¹¹In was determinedby the standard equation:% specificlysis=[(experimental-spontaneous)/(maximum-spontaneous)]×100. Whereindicated, CTL activity was converted to lytic units (LU) as describedby Wunderlich et al, 1994.

Anti-Vaccinia Antibody Analysis

V-WT was added at 5×10⁵/well to polyvinyl chloride plates (Dynatech,Chantilly, Va.), dried overnight at 37° C. and blocked with 5% BSA.Graded dilutions of sera from immunized mice was added in triplicate andincubated for 1 h at 37° C. Plates were washed and incubated withperoxidase labeled goat anti-mouse IgG (Kirkegaard and PerryLaboratories, Gaithersburg, Md.) for an additional hour. Wells weredeveloped with o-phenylenediamine dihydrochloride (Sigma, St. Louis,Mo.) and H₂O₂. Reactions were stopped with H₂SO₄. The absorbance of eachwell was read at 405 nm using a Bio-Tek EL312e microplate ELISA reader(Winooski, Vt.).

Results Increased Expression of Costimulatory Molecules on DC

To determine the efficiency of poxvirus infection of DC, these cellswere infected with either a rV virus encoding B7-1, ICAM-1, and LFA-3(designated rV-TRICOM) or a rF virus encoding B7-1, ICAM-1, LFA-3 andhuman carcinoembryonic antigen (CEA) (designated rF-CEA/TRICOM). In thelatter case, CEA was used as a reporter gene since fowlpox structuralproteins are not expressed in infected cells. After 18 h, cells wereanalyzed for the expression of cell-surface markers associated with theparticular viral infection. Uninfected control DC expressed CD11b (97%)and were negative for the expression of vaccinia proteins. Afterinfection with rV-TRICOM, 94% of DC co-expressed both CD 11b andvaccinia proteins. DC infected with rF-CEA/TRICOM co-expressed bothCD11b and CEA (87%). These DC failed to express fowlpox proteins asdetected by polyclonal rabbit anti-fowlpox sera (data not shown), whichis in agreement with reports stating that fowlpox does not replicate inmammalian cells. Taken together, these data indicate that DC areefficiently infected by both rV and rF vectors.

The cardinal characteristics of DC are high expression levels of bothhistocompatibility antigens and costimulatory molecules. To furthercharacterize the phenotype of DC after virus infection, cells wereinfected with wild-type vaccinia virus (V-WT), rV-B7-1, rV-TRICOM,wild-type fowlpox (FP-WT) or rF-TRICOM and analyzed for the expressionof cell-surface markers associated with the DC phenotype (Table 4). Asexpected, uninfected and mock-infected DC expressed high levels of MHCClass I and II, CD11b, B7-2 and CD40 molecules, as well as high levelsof B7-1, ICAM-1, and LFA-3. DC infected with V-WT expressed lowercell-surface densities (as determined by MFI) of several molecules,while DC infected with rV-B7-1 expressed 5-fold more B7-1 thanuninfected DC (MFI from 329 to 1689). Infection of DC with rV-TRICOMsubstantially increased MFI and the percentage of cells positive forB7-1, ICAM-1, and LFA-3. DC infected with FP-WT had a similar phenotypicprofile to that of uninfected DC. Infection of DC with rF-TRICOM alsosubstantially increased MFI and the percentage of cells positive forB7-1, ICAM-1, and LFA-3. All DC populations remained negative for T-cell(CD3), B-cell (CD19), monocyte/neutrophil (CD14), and NK-cell (Pan NK)markers both before and after infection with rF or N vectors (Table 4).

TABLE 4 Infection of BMDC with rV-TRICOM or FP-TRICOM Increases theExpression Level of B7-1, ICAM-1, and LFA-3 DC Panel [% positive cells(MFI)] Infection I-A^(b) H-2K^(b)/D^(b) CD11b CD11c B7-2 CD40 B7-1 None88 (1124) 89 (125) 93 (935) 20 (74) 68 (490) 68 (82) 91 (329) Mock 87(989) 88 (125) 90 (1129) 25 (49) 77 (432) 71 (99) 85 (330) V-WT 87 (890)86 (83) 86 (588) 29 (54) 61 (274) 79 (98) 90 (197) rV-B7-1 85 (856) 85(104) 81 (693) 25 (62) 73 (304) 73 (103) 94 (1689) rV-TRICOM 87 (901) 78(103) 77 (558) 22 (54) 66 (298) 71 (81) 96 (1442) FP-WT 91 (985) 98(126) 89 (889) 24 (69) 65 (487) 71 (90) 94 (382) rF-B7-1 88 (987) 99(114) 86 (793) 28 (72) 74 (404) 68 (90) 95 (1559) rF-TRICOM 90 (900) 99(115) 84 (789) 27 (66) 76 (499) 73 (93) 99 (1824) DC Panel [% positivecells (MFI)] Non-DC Markers Infection ICAM-1 LFA-3 CD14 CD19 CD3⁺ Pan NKNone 96 (595) 88 (153) 2 (20) 2 (26) 0.3 (33) 2 (56) Mock 97 (519) 86(189) 1 (42) 4 (30) 0.6 (37) 2 (29) V-WT 95 (241) 70 (196) 3 (20) 3 (45)  1 (59) 5 (50) rV-B7-1 97 (364) 72 (131) 3 (42) 3 (40) 0.8 (67) 4 (37)rV-TRICOM 94 (1528) 92 (304) 3 (32) 4 (29) 0.9 (70) 4 (31) FP-WT 97(464) 91 (130) 2 (18) 2 (10)   1 (60) 2 (31) rF-B7-1 98 (437) 90 (180) 2(30) 2 (29)   1 (45) 2 (54) rF-TRICOM 98 (1697) 95 (530) 3 (22) 3 (13)  1 (50) 3 (33) DC were uninfected, mock infected, or infected with 25MOI of either V-WT, rV-B7, or rV-TRICOM or 50 MOI of either WT-FP orFP-TRICOM for 5 h. After 18 h incubation, cells were phenotyped by3-color flow cytometric analysis. Bold numbers indicate a > 100% changein cell-surface expression (MFI).DC Infected with TRICOM Vectors Exhibit Enhanced Capacity to StimulateNaïve T Cells

An in vitro model was used to analyze how increased levels of B7-1,ICAM-1 and LFA-3 expression help induce naïve T-cell proliferation. Inthis model, the first signal for T-cell activation was delivered via apharmacological reagent (Con A) and the additional, or costimulatory,signal was delivered to the T-cell via DC or DC expressing higher levelsof TRICOM as a consequence of recombinant poxvirus infection. In theseand all subsequent studies reported here, V-WT and FP-WT were also usedto rule out effects due to the vector alone. As shown in FIG. 36A, bothuninfected and mock-infected DC induced proliferation of T-cells. DCinfected with V-WT (designated DC/V-TRICOM) induced less T-cellproliferation than uninfected DC. Delivery of additional costimulatorysignals via DC infected with rV-B7-1 (designated DC/rV-B7-1) increasedproliferation compared with uninfected DC. However, DC infected withrV-TRICOM (designated DC/rV-TRICOM) induced further increases in T-cellproliferation at all concentrations of Con A. In addition, when T-cellswere stimulated with DC/rV-TRICOM, 28-fold less Con A was needed toinduce proliferation to levels comparable to that of uninfected DC. Whenthese experiments were repeated using fowlpox vectors, DC/rF-TRICOMinduced increases in T-cell proliferation at all Con A concentrations,unlike DC or DC/rF-B7-1 (FIG. 36B). These experiments were repeated 4times with similar results.

Enhanced Allostimulatory Activity by DC Infected with TRICOM Vectors

The effect of rV-TRICOM (FIG. 37A, C, E) or rF-TRICOM (FIG. 37B, D, E)infection on DC stimulatory capacity was assessed in an allospecificmixed-lymphocyte reaction. Both uninfected DC and mock-infected DCpopulations induced a strong proliferation (78,000 CPM) of allogeneic Tcells (FIG. 37A, B). The stimulatory capacity of DC was increased afterinfection with rV-B7-1 (FIG. 37C). Infection of DC with rV-TRICOMincreased the stimulatory capacity over DC and DC/rV-B7-1 at allDC/responder ratios (FIG. 37C). Importantly, DC populations infectedwith rV-TRICOM vectors failed to stimulate syngeneic T cells (FIG. 37E).When these experiments were repeated using fowlpox vectors (FIG. 37B,D), DC/rF-TRICOM induced larger increases in allogeneic T-cellproliferation than DC and DC/rF-B7-1, DC/rF-TRICOM, however, failed tostimulate syngeneic T cells (FIG. 37F). These experiments were repeated3 times with similar results.

In Vitro Costimulation Analysis: Presentation of Peptides to Effector TCells

Studies were undertaken to determine if the stimulatory capacity ofpeptide-pulsed DC could be enhanced by infecting DC with rV-TRICOM. Tothat end, the H-2K^(b)-restricted OVA (ovalburnin₂₅₇₋₂₆₄, SIINFEKL)peptide and an OVA-specific CD8⁺ effector T-cell line were used. DC wereexposed to different concentrations of OVA peptide and incubated in thepresence of the OVA T-cell line (FIG. 38A-38F). The conventional (i.e.,uninfected) DC induced a strong proliferation of OVA-specific T cellswhen incubated with the OVA peptide (FIG. 38A). These DC did not induceproliferation of OVA-specific T cells when incubated with the controlpeptide VSVN (vesicular stomatitis virus N₅₂₋₅₉ RGYVYQGL) (FIG. 38A,open squares). DC/rV-B7-1 increased the overall peptide-specificproliferation of these cells 1.8-fold (FIG. 38C). In addition,DC/rV-B7-1 induced similar proliferation to that of uninfected ormock-infected DC in the presence of 4-fold less peptide. In contrast,DC/rV-TRICOM increased the overall proliferation of these T-cellsseveral-fold, and in the presence of 32-fold less OVA peptide, inducedproliferation comparable to that of uninfected DC (FIG. 38C). To furtherevaluate the capacity of vaccinia-infected DC to present peptide, DCwere pulsed with a single concentration of OVA peptide (1 μM) andincubated in the presence of several ratios of T cells (FIG. 38E). On aper-cell basis, 4-fold fewer DC/rV-B7-1 were required to induceproliferation levels comparable to that of DC (open triangles vs. closedsquares). The greatest stimulatory effect was that of DC/rV-TRICOM,which induced proliferation levels comparable to that of DC with 32-foldless cells (open circles vs. closed squares).

A second peptide system employing peptide-pulsed DC and an establishedT-cell line were employed to determine if results similar to thoseobtained with the OVA peptide could be noted. These experiments wereconducted using the H-2D^(b)-restricted peptide CAP-M8 (CEA₅₂₆₋₅₃₃,EAQNTTYL) and a CAP-M8-specific CD8⁺ effector T-cell line; similarresults were noted (FIG. 38B, D, F). These experiments were repeated 5additional times with the same results.

Effect of rV-TRICOM Infection on TNFα or CD40-Matured DC

Since the functional maturation of DC is believed to correlate with theupregulation of T-cell costimulatory molecules, experiments wereconducted to examine the effect of rV-TRICOM infection on DC that hadbeen matured by co-culture with either TNF-α or CD40 mAb. Treatment ofDC with TNF-α during the final 24 h of culture resulted in someupregulation of MHC-II, B7-2, and ICAM-1 as determined by flowcytometric analysis (Table 5), while treatment of DC with CD40 mAbresulted in the upregulation of ICAM-1 expression and a slightupregulation of MHC-II. Functionally, treatment of DC with TNF-αc orCD40 mAb culminated in a 28% and 16% increase, respectively, inpeptide-specific proliferation over that of unmanipulated DC (FIG. 39A).Similar data were also obtained after treating DC withlipopolysaccharide (LPS). Infection of untreated DC with rV-TRICOMresulted in a substantial increase in T-cell proliferation (FIG. 39A vs.39B). Pretreatment with TNF-α or CD40 mAb followed by infection withrV-TRICOM, however, conferred only a slight stimulatory capacity inexcess of that seen with rV-TRICOM infection alone (FIG. 39B). Theseexperiments were repeated 3 additional times with similar results.

TABLE 5 Effect of Pretreatment of DC with TNF-α or CD40 mAb Prior torV-TRICOM Infection Pre- DC Panel [% positive cells (MFI)] Infectiontreatment I-A^(b) H-2K^(b)/D^(b) CD11b CD11c B7-2 CD40 B7-1 ICAM-1 LFA-3DC (Uninfected) None 90 (924) 93 (225) 90 (835) 26 (174) 65 (340) 62(182) 93 (389) 96 (415) 90 (253) DC (Uninfected) TNF-α 95 (1189) 91(195) 84 (729) 20 (149) 71 (412) 65 (159) 85 (320) 97 (421) 87 (249) DC(Uninfected) CD40 91 (990) 89 (183) 87 (788) 22 (154) 68 (374) 69 (198)90 (297) 95 (690) 86 (216) mAb DC/rV-TRICOM None 87 (756) 89 (214) 85(684) 24 (98) 69 (301) 66 (103) 95 (1989) 98 (1487) 93 (413)DC/rV-TRICOM TNF-α 92 (991) 90 (230) 79 (558) 21 (62) 72 (398) 68 (81)96 (1442) 94 (1998) 90 (394) DC/rV-TRICOM CD40 91 (905) 90 (216) 81(614) 23 (69) 65 (387) 71 (120) 94 (1382) 97 (1444) 89 (310) mAb DC weretreated with TNF-α (100 ng/ml) or CD40 mAb (1 μg/ml) during the final 24h of culture. DC or treated DC were then infected with 25 MOI ofrV-TRICOM for 5 h. After 18 h incubation, cells were phenotyped by3-color flow cytometric analysis.DC Infected with W-TRICOM Are More Efficient at Priming CTL Responses InVivo

Experiments were conducted to determine if the enhanced stimulatorycapacity of DC/rV-TRICOM noted in vitro using Con A (FIG. 36E-F),mixed-lymphocyte reactions (FIG. 37) and two effector T-cell models(FIG. 38) would translate to enhanced efficacy in priming naive T-cellresponses in vivo. To that end, DC, DC/V-WT, and DC/rV-TRICOM werepulsed with 10 μM OVA peptide and administered intravenously to C57BL/6mice. Control mice were immunized with OVA peptide in Ribi/Detoxadjuvant subcutaneously. Splenocytes were harvested 14 days followingvaccination, restimulated in vitro for 6 days, and assessed for theirpeptide-specific lytic ability against OVA-pulsed EL-4 cells. EL-4 cellspulsed with VSVN peptide were used as control target cells. As seen inFIG. 40A, CTL generated from mice immunized with peptide/adjuvantexhibited modest levels of CTL activity (FIG. 40A). Mice immunized withpeptide-pulsed DC exhibited a greater peptide-specific CTL response(FIG. 40B). The induced CTL response was somewhat blunted in miceimmunized with DC/v-WT (FIG. 40C, <2.5 lytic units (LU) vs. 5.2 LU). Incontrast, mice immunized with peptide-pulsed DC/rV-TRICOM (FIG. 40D)exhibited a CTL response that was significantly stronger than that of DC(LU=14.3, p=0.001). Similar experiments were then conducted using asecond model peptide, CEA peptide CAP-M8 (FIG. 40E-H). Again,peptide-pulsed DC elicited much greater CTL activity than that educed bypeptide/adjuvant (5.7 LU vs. <2.5 LU). In addition, mice immunized withpeptide-pulsed DC/rV-TRICOM (FIG. 40H) exhibited a strong CTL response(>20 LU) compared with that induced by peptide-pulsed DC (5.7 LU,p=<0.001; FIG. 40F).

Efficacy of Multiple Vector-Infected DC Vaccinations

It is generally believed that the generation of anti-vaccinia antibodiescan prevent the repeated use of vaccinia virus as immunogens. However,little is known about the repeated use of vaccinia-infected cells asimmunogen. To address this issue, an immunization scheme was carried outin which CAP-M8 peptide-pulsed DC immunogens were administered one, two,or three times, at 7-day intervals. As before, splenocytes wereharvested 14 days following the final immunization, restimulated invitro for 6 days, and assessed for their peptide-specific lytic abilityagainst CAP-M8-pulsed EL-4 cells. As seen in FIG. 41A, peptide-pulsedDC/rV-TRICOM induced higher levels of CTL activity when compared withpeptide-pulsed DC. These data are similar to those seen in FIG. 40E-H.This single administration of DCN-WT or DC/rV-TRICOM induced significantanti-vaccinia IgG antibody titers, with values ranging from 1:4,000 to1:9,000 as determined by qualitative ELISA. These titers, however, hadno effect on the capacity of these immunogens to boost CTL activity uponsubsequent immunizations (FIGS. 41B and 41C). While anti-vaccinia virustiters after the second vaccination ranged from 1:12,000 to 1:50,000, aboost in the induction of peptide-specific CTL was seen in all groups.Again, the CTL activity observed employing DC/rV-TRICOM-pulsed cells wasgreater than that observed with peptide-pulsed DC.

EXAMPLE 32 Splenocytes or Bone Marrow Progenitor Cells Infected WithTRICOM Vectors Induce T-cell Activation Comparable to Dendritic CellsMaterials and Methods Generation of Bone Marrow Progenitor Cells andDendritic Cell Cultures

The procedure used for generation of bone marrow-derived DC was thatdescribed by Inaba et. al. with minor modifications. Briefly, the femurswere taken from 6-8 week old female C57BU6 mice (Taconic Farms,Germantown, N.Y.) and the bone marrow was flushed and passed over aFicoll-Hypaque gradient. Bone marrow cells were depleted of lymphocytesand Ia⁺ cells using a cocktail of magnetic beads specific for CD4, CD8,and MHC Class II (MiniMACS, Miltenyi Biotec, Auburn, Calif.). Designatedas dendritic cell progenitors, these depleted bone marrow cells werethen prepared for infection, or for dendritic cell cultures depletedbone marrow cells were plated in six-well culture plates (10⁶ cells/ml,5 ml/well) in CM supplemented with 10 ng/ml GM-CSF and 10 ng/ml IL-4(R&D Systems, Minneapolis, Minn.). DC cultures were replated in freshcytokine-supplemented CM on days 2 and 4, and split to new plates on day4. At day 7 of culture, cells were harvested for analysis, in vitroassays, and in vivo immunizations.

Generation of Splenocyte Stimulator Cells.

Spleens were harvested from naïve female C57BL/6 mice, crushed into asingle-cell suspension, and passed over a Ficoll-Hypaque gradient.Splenocytes were depleted of lymphocytes and Ia⁺ cells using a cocktailof magnetic beads specific for CD90, and MHC Class II. Purifiedsplenocytes were then washed twice with Opti-Mem (Gibco-BRL) andprepared for infection with the recombinant poxviruses.

Infection of Stimulator Cells.

Bone marrow-derived dendritic cell progenitor and splenocyte cells werewashed twice with Opti-Mem and mock infected or infected with either 25MOI V-WT, rV-B7-1, rV-TRICOM, or 50 MOI FP-WT, rF-B7-1 or rF-TRICOM at25 MOI (multiplicity of infection, PFU/cell) in 1 ml final volume ofOpti-Mem for 5 hours. After infection, warm (37 degree) CM was added andthe cells were incubated at 37° C. overnight. After infection the cellswere harvested for immunostaining, in vitro costimulation analysis, andin vivo administration.

Costimulation Analysis

Rested CAP-M8 T-cells (responders) were added at 5×10⁴/well in a 96-wellflat-bottomed plates (Costar, Cambridge, Mass.). Stimulator cellsconsisted of BMDC, splenocytes, or bone marrow progenitors, eitheruninfected, mock infected, or infected with either V-WT, rV-B7-1,rV-TRICOM, FP-WT, or rF-TRICOM and irradiated (20 Gy). Cells in wellswere cultured in a total volume of 200 ml of CM. The costimulation assaywas carried out using two sets of conditions: a) fixed ratio ofresponder:stimulator cell of 2.5:1 for non-BMDC stimulators, and 10:1for BMDC, cultured in the presence of several concentrations of Con-A assignal one, specific peptide, or appropriate control peptide, or b) afixed concentration of Con-A as signal one, specific peptide, or controlpeptide, cultured at various responder:stimulator cell ratios. Cellswere cultured for 48 or 72 hours for Con-A and peptide-specific assays,respectively, and labeled for the final 12-18 hours of the incubationwith 1 mCi/well 3H-Thymidine, harvested, and analyzed as describedabove.

Table 6 shows splenocyte and bone marrow (BM) cell surface expression ofcostimulatory molecules after infection with recombinant vectors.Purified murine splenocytes or bone marrow cells were infected for 5hours with 25 MOI of vaccinia vectors or 50 MOI of fowlpox vectors. Cellphenotype was compared with that of DC. All cells were immunostainedwith costimulatory molecule-specific mAbs labeled with fluoresceinisothliocyanate, phycoerythrin, or biotin/streptavidin-cychrome. Isotypecontrol were negative (data no shown). Numbers indicate percent positivecells and mean fluorescence intensity in parentheses.

TABLE 6 Infection of BMDC, Splenocytes, and BMDC Progenitors with eitherrV-Tricom or rF- TRICOM increases the level of expression of B7-1,ICAM-1, and LFA-3¹ I-A^(b) H-2K^(b)/D^(b) CD11b CD11c CD40 B7-2 B7-1ICAM-1 LFA-3 CD19 DC² Uninfected  88 (1124) 89 (125) 93 (935) 20 (74) 68 (82)   91 (329) 91 (329) 96 (595)  88 (153)  2 (26) Spleno-Uninfected 92 (102) 96 (389)  3 (136) 1 (54)  87 (494) 49 (61) 46 (540)85 (258) 77 (40) 42 (25) cytes³ V-WT 91 (114) 94 (400)  3 (182) 0.7(82)   75 (408) 63 (92) 55 (490) 76 (257) 47 (32) 25 (24) rV-B7 91 (123)95 (402) 3 (89) 1 (159) 81 (369) 61 (89)  87 (1134) 85 (315) 45 (29) 33(27) rV-Tricom 93 (188) 98 (433) 3 (41) 3 (83)  81 (327) 49 (69)  87(1104) 92 (788)  97 (192) 27 (33) FP-WT 90 (104) 90 (410)  2 (162) 0.9(92)   79 (418) 60 (72) 55 (460) 70 (157) 49 (32) 53 (29) rF-B7-1 91(133) 86 (422) 1 (81) 1 (149) 85 (399) 55 (96) 83 (830) 83 (215) 51 (29)52 (31) rF-Tricom 89 (238) 96 (399) 3 (91) 2 (80)  86 (387) 51 (99)  86(1001) 92 (588)  99 (292) 48 (33) BM⁴ Uninfected  9 (289) 99 (389) 80(114) 1 (909) 26 (136) 28 (72) 79 (115) 68 (144) 37 (89)  1 (147) V-WT 8 (218) 98 (236) 66 (144)  1 (1131) 19 (161) 19 (98) 75 (131) 63 (151)33 (64)  5 (50) rV-B7  8 (192) 97 (159) 71 (144) 2 (394) 25 (233)  22(125)  89 (1117) 56 (204) 31 (65)  2 (106) rV-Tricom  7 (242) 92 (183)70 (129) 1 (875) 16 (171) 16 (91) 92 (880) 80 (490)  38 (112)  3 (62)FP-WT  8 (318) 98 (298) 64 (133)  1 (1101) 23 (175) 22 (88) 74 (121) 60(112) 35 (69)  2 (30) rF-B7-1  7 (292) 99 (259) 75 (129)  2 (1001) 26(245)  26 (101) 91 (652) 50 (104) 34 (72)  1 (86) rF-Tricom  8 (233) 96(213) 72 (118) 1 (984) 25 (111) 13 (99)  96 (1880) 79 (310)  39 (109)  1(52) ¹Cells were uninfected, or infected with 25 MOI of V-WT, rV-B7-1,rV-Tricom or 50 MOI FP-WT, rF-B7-1, or rF-TRICOM for five hours. Afteran eighteen hour incubation period, cells were phenotyped by 3-colorflow cytometric analysis. Values are given as [% positive cells (MeanFlourescence intensity)]. Bold numbers indicate a >2-fold change in cellsurface expression (MFI). ²BMDC: day 6 bone marrow derived dendriticcells (cultured in 10 ng/ml GM-CSF/IL-4). ³Splenocytes depleted ofT-cells via α-CD90 (Thy 1.2) magnetic beads. ⁴

indicates data missing or illegible when filed

FIGS. 42A through 46 demonstrate that TRICOM-infected splenocytes arecomparable to TRICOM infected bone marrow cells in stimulating T cellresponses.

EXAMPLE 33 Human T cell Stimulation Using Allogeneic rF-TRICOM InfectedHuman Dendritic Cells Pulsed with Peptides

Human dendritic cells were isolated for a normal, healthy individual byleucophoresis. The human dendritic cells were cultured in the presenceof GM-CSF and IL-4 for 6-9 days, followed by the addition of rF-TRICOMor rF-Controls for infection of the dendritic cells. TherF-TRICOM-infected dendritic cells were pulsed with a CEA peptide (CAP-1or CAP1, 6D) (FIG. 47); a PSA peptide (PSA-3) (FIG. 48); an influenzapeptide (Flu peptide 58-66) (FIGS. 49 and 50); or an HPV peptide (11-20)(FIGS. 51-45) for 1 hour. Human T cells isolated from peripheral bloodmononuclear cells (PBMC) were cultured in the presence of thepeptide-pulsed rF-TRICOM-infected dendritic cells and production ofIFN-α by the T cells determined. FIGS. 47-54 show that peptide-pulsedrF-TRICOM infected human dendritic cells stimulated T cells to a greaterextent than the controls. FIGS. 47-54, as well as Table 7 demonstratethat allogeneic human dendritic cells infected with rF-TRICOM canefficiently present any antigenic peptide to T cells for enhancement ofan immune response.

TABLE 7 CTL activity of T cell lines by using DC pulsed with HPVE7(11-20) pepride T cell lines established by using A B TargetrF-Tricom + P rF-B7.1 + P rF-FPV + P DC + P C1R-A2 + 39.6 (3.1) 24.7(0.4) 19.9 (2.9) 7.3 (0.4) HPV C1R-A2  5.1 (2.0)  6.9 (4.0)  7.6 (2.0)8.0 (0.2) E:T ratio = 25:1 An 6 hour 111-In release assay was performed.C1R-A2 cells were pulsed with HPV E7 peptide (11-20) YMDLQPETT at aconcentration of 10 μg/ml.

The results presented in Table 7 demonstrate that DC infected withrF-TRICOM (A), are better as APC to generate CTL than are standard DC(B) when both are pulsed with peptide.

EXAMPLE 34 Human Clinical Trials of a rV-huTRICOM, rV-CEA huTRICOMVaccine and rF-CEA TRICOM

The objective of the human clinical trial is to determine the optimumtolerated dose (OTD) of the recombinant rV-huTRICOM and rV-CEA-huTRICOMvaccine that elicits a host anti-tumor immune response and is associatedwith acceptable toxicity in patients with advanced CEA-expressingadenocarcinomas.

The rV-huTRICOM and rV-CEA-huTRICOM vaccines are produced underconditions suitable for Phase I and Phase II human clinical trial.

In an initial trial, escalating doses of recombinant rV or rFCEA-huTRICOM live virus vaccine or rV-huTRICOM plus rV-CEA vaccine isprovided at an initial dose of 10⁶ pfu virus, I.M., followed by a doseof 10⁷ pfu virus, I.M., which is followed later by of 10⁸ pfu virus, or10⁹ S.C. or by scarification.

The anti-tumor response to each recombinant vaccine is determined usingclinical, laboratory and radiologic evidence of tumor size, extent andgrowth using accepted standard criteria for measuring response of tumorsto new forms of therapy as are known in the art.

The patient's immune response to the recombinant vaccine is assessedusing a variety of immunological assays including anti-CEA antibodyassay, anti-poxvirus antibody assay, immune complex assay, CEA-specificlymphoproliferative assay, CEA-specific cytotoxic T-lymphocyte assays,precursor frequency of CEA-reactive T cells in gamma-interferon releaseT-cell assay, a ELISPOT, Fast Immune, Tetramere assays for T-cellresponses (Scheibenhogen et al Int. J. Cancer 71:932-936, 1997), HLAassays and the like. A comparison of pre-treatment and post-treatmentsamples are made to document development of humoral and cellular immuneresponses directed against the CEA tumor antigen.

EXAMPLE 35 Human Clinical Trials of an Recombinant Fowlpox-CEA-huTRICOM

In an initial trail, escalating doses of recombinant fowlpox-CEAhuTRICOM vaccine of 10⁶ pfu virus, 10⁷ pfu virus and 10⁸ pfu virus isinjected directly into a tumor mass of a patient with advancedCEA-expressing adeno carcinomas.

The specific anti-tumor and immune response to the recombinant vaccineis determined as described in Example 34.

EXAMPLE 36 Human Clinical Trial of T Lymphocytes Activated by MultipleCostimulatory Molecule-Overexpressing Dendritic Cells

Peripheral blood lymphocytes and dendritic cells are obtained from apatient with advanced prostate cancer. The peripheral blood lymphocytesare enriched for CD8+ lymphocytes. The dendritic cells are infected withrV-PSA epitope QVHPQKVTK/B7.1/ICAM-11/LFA-3 for a period of timesufficient to allow expression of the PSA epitope and overexpression ofthe multiple costimulatory molecules. PSA epitope-specific CD8′lymphocytes are activated and expanded in the presence of these treateddendritic cells. The activated PSA epitope-specific CD8⁺ autologous Tlymphocytes are injected into the patient alone and in combination withthe PSA epitope. The specific anti-tumor and PSA-specific immuneresponse to the treatment is determined by methods comparable to thosedescribed in Example 34.

Similar human clinical trials may be conducted for treatment of patientswith other TAA-expressing cancers, by replacement of the gene encodingCEA with a gene encoding another TAA into the recombinant vector of thepresent invention.

EXAMPLE 37 Screen for Immunogenic Peptides and/or Human T CellsImmunoreactive with a Specific Peptide Using DC Infected with rF-TRICOM

The present invention encompasses anticancer therapies using ex vivoengineering of DC with viral vectors carrying a tumor associated antigengene to activate tumor-specific CTL. DC infected with rF-CEA incombination with TRICOM costimulatory molecules are used to augmentCEA-specific immune responses. The CTL induction capacity of DC infectedwith rF-CEA/TRICOM and rF-TRICOM are evaluated. Tetrameric MHC class ICAP-1 complex are used to visualize CAP-1 specific CTL. This protocol isnot limited to the tumor associate antigen, CEA, but may be modified toelicit antigen-specific immune responses for any antigenic peptide orimmunogenic epitope thereof for immunotherapy against cancer, pathogenicbacteria, virus, protozoans, yeast and the like. Moreover, the methodmay be modified to screen for and identify immunogenic peptides from asource such as a natural protein, recombinant protein, syntheticprotein, or fragments from each, combinatorial libraries, and the like.

Materials and Methods Cell Cultures

Colorectal carcinoma cell lines SW1463 (HLA-A1,2), LS174T (HLA-A2,-),were purchased from American Type Culture Collection (Manassas, Md.).The cultures were free of mycoplasma and were maintained in completemedium [DMEM (Life Technologies, Inc., Grand Island, N.Y.) supplementedwith 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin,and 100 μg/ml streptomycin (Life Technologies, Inc.)]. The CIR cell lineis a human plasma leukemia cell line that does not express endogenousHLA-A or B antigens (Storkus, W. J. et al, J. Immunol. 138(6):1657-1659,1987). C1R-A2 cells are C1R cells that express a transfected genomicclone of HLA-A2.1 (Hogan, K. T. et al, J. Exp. Med. 168(2):725-736,1988). These cells were obtained from Dr. William E. Biddison (NationalInstitute of Neurological Disorders and Stroke, NIH, Bethesda, Md.).C1R-A2 culture was mycoplasma free and was maintained in RPMI 1640complete medium (Life Technologies, Inc.). The V8T cell line, a CTL linedirected against the CAP-1 epitope, was established from a patient withmetastatic colon carcinoma who was enrolled in a Phase I trial usingrV-CEA (Tsang, K. Y. et al., Clin. Cancer Res. 3(12):2439-2449, 1997).V8T cells were cultured in RPMI 1640 complete medium containing 10%human AB serum and IL-2 (provided by the National Cancer Institute,Surgery Branch, 20 units/ml). V8T cells were restimulated with CAP-1peptide (25 μg/ml) on day 16 after prior restimulation at an effectorcell-to-APC ratio of 1:3. Irradiated (23,000 rads) autologous EBVtransformed B cells were used as APC.

Culture of DC from Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMC) were obtained from heparinizedblood from a patient (#15) with metastatic pelvic carcinoma who wasenrolled in a Phase I trial using a combination of rV-CEA and ALVAC-CEA.All experiments involving patient materials were conducted according toNIH guidelines, and written, informed consent was obtained from allindividuals. PBMC were separated using lymphocyte separation mediumgradient (Organon Teknika, Durham, N.C.) as described previously (Boyum,A. Scand J Clin Lab Invest Suppl. 97:51-76, 1968). DC were preparedusing a modification of the procedure described by Sallusto et al.(Sallusto, F. et al, J. Exp. Med. 179(4):1109-1118, 1994). PBMC(1.5×10⁸) were resuspended in AIM-V medium containing 2 mM glutamine, 50μg/ml streptomycin, 10 μg/ml gentamycin (Life Technologies, Inc.) andallowed to adhere to a T-150 flask (Corning Costar Corp., Cambridge,Mass.). After 2 hours at 37° C., the non-adherent cells were removedwith a gentle rinse. The adherent cells were cultured for 6-7 days inAIM-V medium containing 50 ng/ml of recombinant human GM-CSF (rhGM-CSF)and 0.5 ng/ml of recombinant human IL-4 (rhIL-4). The culture medium wasreplenished every three days.

Recombinant Virus and Infection of DC with Avipox Virus Containing CEA,CEA/TRICOM and TRICOM

A 2109 bp DNA fragment encoding the entire open reading frame of CEA wasobtained as described by Kaufman et al (Kaufman, F. et al. Int. J.Cancer 48(6):900-907, 1991). The recombinant CEA avipox virus (fowlpoxCEA; vCP248) was supplied by Therion Corp using methods described byTaylor et al (Taylor, J. et al, Virology 187(1):321-328, 1992), Cox etal (Cox, W. I. et al, Virology 187(1):321-328, 1992) and Perkus et al(Perkus, M. E. et al, J. Virol. 63(9):3829-3836). The recombinant avipoxvirus encoding CEA and human Tricom gene (designated rF-CEA-Tricom) andthe recombinant human fowlpox-TRICOM (rF-Tricom) were made as disclosedherein. Wild type fowlpox (FP-WT) was used as a negative control inselected experiments. DC (1×10⁶) were incubated in 1 ml of Optim-MEMmedium (Life Technologies, Inc.) at 37° C. with rF TRICOM, rF-CEA,rF-CEA/TRICOM, FP-WT. Titration experiments indicated that 2×10⁷plaque-forming units/ml, equal to a multiplicity of infection (MOI) of40:1 for 2 hours, were able to consistently induce expression of CEA inapproximately 75% of the infected DC. The infected DC were suspended in10 ml of fresh, warm RPMI-1640 complete medium containing 50 ng/ml ofrhGM-CSF and 0.5 ng/ml rhIL-4 cultured for 24 hours, and thensubsequently used as stimulators.

Peptide

CAP-1 (Tsang, K. Y. et al, J. Natl Cancer Inst. 87(13):982-990, 1995),CEA amino acid position 571-579 YLSGANLNL, CAP1-6D (Zaremba, S. et al,Cancer Res. 57(20):4570-4577, 1997) YLSGADLNL and Flu peptide, influenzamatrix protein peptide 58-66 GILGFVTL greater than 96% pure, were madeby Multiple Peptide System (San Diego, Calif.).

Generation of T-Cell Lines

Modification of the protocol described by Tsang et al (Tsang, K. Y. etal, J. Natl Cancer Inst. 87(13):982-990, 1995) was used to generateCEA-specific CTL. Uninfected DC and DC infected with rF-TRICOM, rF-CEA,or rF-CEA/TRICOM were used as APC. CAP-1 peptide was added to theuninfected or rF-TRICOM infected DC at a final concentration of 25μg/ml. Autologous non adherent cells were then added to APC at anAPC-to-effector ratio of 1:10. Cultures were then incubated for 3 daysat 37° C. in a humidified atmosphere containing 5% CO₂. After removal ofthe peptide-containing medium, the cultures were then supplemented withrecombinant human IL-2 at a concentration of 20 units/ml for 7 days,with IL-2 containing medium was replenished every 3 days. The 3-dayincubation with peptide and 7 day IL-2 supplement constituted one IVScycle. Primary cultures were restimulated with CAP-1 peptide (25 μg/ml)on day 11 to begin the next IVS cycle. Irradiated (23,000 rads)autologous EBV-transformed B cells were used as APC. A similar procedurewas employed for CTL generation when DC infected with rF-CEA orrF-CEA/TRICOM were used as APC, with the exception that no CAP-1 peptidewas in the stimulation.

Construction of Peptide MHC Tetramers

Peptide-MHC complexes were synthesized as described by Altman et al(Altman, J. D. et al Science 274(5284):94-95, 1996). In brief, the β₂microglobulin (β₂M) clone was obtained from Dr. Garboczi (HarvardUniversity, Cambridge, Mass.) (Garboczi, D. N. et al., Proc Natl AcadSci USA 89(8):3429-3433, 1992) and the HLA-A2 construct was obtainedfrom Immunotech (Beckman-Coulter, Marseille, France). The soluble HLA-A2molecules containing the 15 amino acid substrate peptide forBirA-dependent biotinylation to the COOH-terminus of the HLA-A2 heavychain and β₂M were grown separately in E. coli and isolated as inclusionbodies. HLA-A2 and β₂M were solubilized and renatured in the presence ofCAP-1 or Flu-M158-66 peptide. The complex was purified by FPLC onSuperdex 200 (Pharmacia, Piscataway, N.J.). Purified peptide-MHC complexwas biotinylated using the BirA enzyme (Avidity, Denver, Colo.).Tetramers were produced by mixing the biotinylated peptide-MHC complexwith phycoerythrin-labeled UltraAvidin (Leinco Technologies, Inc.Ballwin, Mo.) at a molar ratio of 4:1.

Flow Cytometry

Staining and sorting of T-cells: CAP-1-MHC tetramer-PE was used for flowcytometric analysis and sorting of T-cells. Similar procedure asdescribed above was used for tetramer staining. CAP-1-MHC tetramer-PEwas used at a concentration of 0.33 μg/2×10⁵ cells. Cells were stainedwith CAP-1 MHC tetramer-PE for 1 hour at 4° C. and then stained withanti-CD8 FITC for an additional hour. Cells were washed and analyzed ona Vantage Cell sorter (Becton Dickinson) or a FACScan (Becton Dickinson)using CellQuest software (Becton Dickinson). Sorter cells were culturedand expanded as described previously. Cells stained with UltrAvidin-PEand Flu-MHC tetramer were used as negative controls.

Cytotoxic Assay

Target cells were labeled with 50 μCi of ¹¹¹Indium-labeled oxyquinoline(Medi-Physics Inc., Arlington, Ill.) for 15 min at room temperature.Target cells (0.3×10⁴) in 100 μl of RPMI-1640 complete medium were addedto each of 96 wells in flat-bottomed assay plates (Corning Costar,Corp.). The labeled target cells were incubated with peptides for 60 minat 37° C. in 5% CO₂ before adding effector cells. No peptide was usedwhen carcinoma cell lines were used as targets. Effector cells weresuspended in 100 μl of RPMI-1640 complete medium supplemented with 10%pooled human AB serum and added to the target cells. The plates werethen incubated at 37° C. in 5% CO₂ for 4 or 16 hours. Supernatant washarvested for gamma counting with the use of harvester frames (Skatron,Inc., Sterling, Va.). Determinations were carried out in triplicate, andstandard deviations were calculated. Specific lysis was calculated withthe use of the following formula (all values in cpm):

${\% \mspace{14mu} {lysis}} = \frac{{{Observed}\mspace{14mu} {release}} - {{Spontaneous}\mspace{14mu} {release} \times 100}}{{{Total}\mspace{14mu} {release}} - {{Spontaneous}\mspace{14mu} {release}}}$

Spontaneous release was determined from wells to which 100 μl ofRPMI-1640 complete medium was added. Total releasable radioactivity wasobtained after treatment of targets with 2.5% Triton x-100.

HLA Typing

The HLA phenotyping was performed by the Blood Bank of the NationalInstitutes of Health using a standard antibody-dependentmicrocytotoxicity assay and a defined panel of anti-HLA antisera. Theclass I phenotypes of V8T cell line and patient #15 were HLA-A2, -; B18(W6), 44 (12, W4) and HLA-A2, 28; B13 (BW4), B51 (BW4); CW6,respectively.

Detection of Cytokine

Supernatant of T cells exposed for 24 hours to DC infected with rF-CEA,rF-CEA/TRICOM or to peptide pulsed uninfected DC and rF-TRICOM-infectedDC in IL-2-free medium at various responder:stimulator ratio werescreened for secretion of IFNγ using an ELISA kit (R&D Systems,Minneapolis, Minn.). The results were expressed in pg/ml.

ELISPOT Assay

A modification of the method described by Scheibenbogen et al(Scheibenbogen, C. et al, Clin Cancer Res 3(2):221-226, 1997) was usedto measure IFNγ production to determine CAP-1 specific T cells. Briefly,96-well Milliliter HA plates (Millipore Corporation, Bedford, Mass.)were coated with 100 μl of capture antibody against human IFNγ at aconcentration of 10 μg/ml. After 24 hours incubation at roomtemperature, plates were blocked for 30 min with RPMI-1640 containing10% human pool AB serum. 1×10⁵ cells to be assayed were added to eachwell. CAP-1-6D-pulsed C1R-A2 cells were added into each well as APC atan effector:APC ratio of 1:3. Unpulsed C1R-A2 cells were used asnegative control. HLA-A2 binding Flu Matrix peptide 58-66 (GILGFVFTL)were also used as control. The responding cells were determined by theuse of a Domino Image Analyzer (Otpomax, Hollis, N.H.).

Statistical Analysis

Statistical analysis of differences between means was done using atwo-tailed t test.

Discussion

When a naïve T cell encounters antigen, several distinct outcomes arepossible including proliferation, cytokine secretion, anddifferentiation into effector cells, as well as inactivation, death, andunresponsiveness (anergy). The predominant outcome under physiologicconditions may be determined by whether appropriate costimulatorysignals are delivered to the responding T cell (26). At least threedistinct molecules normally found on the surface of professional APChave been thought to be capable of providing the signals critical forT-cell activation: B7-1, ICAM-1, and LFA-3. Here, the role ofcostimulatory molecules in naive T-cell activation was examined byutilizing vectors engineered to express either B7-1, ICAM-1, LFA-3, or acombination of all three molecules.

Several groups have investigated the cooperation of two of thesemolecules in T-cell costimulation. Dubey et al. have reported thatcostimulation by both B7-1 and ICAM-1 is a prerequisite for naive T-cellactivation (26), while Cavallo et al. determined that B7-1 and ICAM-1must by coexpressed by tumor cells to establish an antitumor memoryresponse (27). In addition, costimulation by B7-1 and LFA-3 has beenshown to act additively both upon T-cell proliferation and cytokineproduction (6, 23, 24). These previous studies were carried out usingtwo costimulatory molecules and retroviral vectors. One gene wastransduced into the target cell line, drug selected, and then transducedagain with a second recombinant retroviral construct followed byselection with a different agent. This process often requires weeks ormonths. Utilizing recombinant poxvirus vectors, one is able to achievethe coexpression of three costimulatory molecules 5 hourspost-infection. In vitro MC38 cells infected with eitherrV-B7-1/ICAM-1/LFA-3 or rF-CEA/B7-1/ICAM-1/LFA-3 were shown to enhanceproliferation of T cells to a much greater extent than MC38 cellsinfected with vectors containing the gene for any single costimulatorymolecule. In addition, the relative strength of the second signaldelivered to the T cell by the combination of costimulatory moleculesappeared to be several-fold (>6) greater than that delivered by MC38cells expressing any single costimulatory molecule. Dubey et al. havedemonstrated that at low stimulator to T-cell ratios, moderate to strongsynergy was noted with B7-1 and ICAM-1 (26). Our studies confirm thesefindings. However, at very low stimulator cell to T-cell ratios or weaksignal-1 (0.625 μg/ml Con A), the two-gene construct (rV-B7-1/ICAM-1)had little if any effect on proliferation; in contrast, stimulation viathe triad construct (rV-B7-1/ICAM-1/LFA-3) had a substantial andstatistically significant effect on proliferation. The predominanteffect of stimulation via the multi-gene construct (rV-B7-1, ICAM-1,LFA-3) was IL-2 elaboration from CD4⁺ cells and IFN-γ elaboration fromCD8⁺ T cells, while few, if any, type 2 cytokines were produced.Cytokine expression analysis by RNAse protection provided a profilecompatible with the in vitro cytokine assay, manifested by significantlyhigher expression of IL-2 and IFN-γ in both CD4⁺ and CD8⁺ T cellsstimulated with all three costimulatory molecules, as compared tostimulation by any single costimulatory molecule. These data are inaccordance with previous studies which demonstrated that in the contextof low CD28 costimulation, T cells produced low levels of IL-1, whereasstrong CD28 costimulation supported production of IL-2, IFN-γ and IL-13(28). Furthermore, it has been reported that IL-13 synergizes with IL-2in regulating IFN-γ synthesis in T cells (29). Interestingly, ourresults further support this observation in that stimulation of CD4⁺ Tcells with MC38/B7-1/ICAM-1/LFA-3 results in a high level of IL-2 andIFN-γ expression, with some increased expression of IL-13. Moreover, itwas noted that IL-9 expression was further enhanced in CD4⁺ T cells uponstimulation with MC38/B7-1/ICAM-1/LFA-3. The increased expression ofIL-9 in conjunction with upregulation of IL-2 noted in our studies is inagreement with previous studies which demonstrated that optimalproduction of IL-9 is regulated by IL-2 (30). Taken together, thesestudies suggest that optimal naive T-cell responses require a higherlevel of costimulation than was previously thought, and that this couldbe provided by the combined action of three costimulatory molecules.

Perhaps the most studied T-cell costimulatory molecule is B7-1. Thismolecule's ability to enhance T-cell activation using retroviralvectors, anti-CTLA-4 antibodies, and poxvirus vectors is wellestablished. The studies reported here rank the order of T-cellstimulation by a single costimulatory molecule as B7-1>ICAM-1>LFA-3.However, the employment of three costimulatory molecules was farsuperior to B7-1 alone or in B7 in combination with a secondcostimulatory molecule in both T-cell proliferation and cytokineproduction.

While not being bound by theory, there are several possible mechanismsfor efficient cooperation between B7-1, ICAM-1 and LFA-3. TheICAM-1/LFA-3 interaction reportedly costimulates the TCR-mediatedactivation of T cells by sustaining the increase in the sameintracellular second messengers as generated by TCR engagement. Thisobservation suggests that the ligation of LFA-1 by ICAM-1 costimulates Tcells by enhancing the signal delivered via the CD3/TCR complex (6). TheICAM-1/LFA-1 interaction is necessary to upregulate expression of theIL-2R-alpha chain and CD28 on T cells, which is required to render themcompetent to respond to IL-2 and B7-1 costimulation. On the other hand,the B7-1/CD28 interaction delivers a TCR-independent costimulatorysignal that increases both transcriptionally and post-transcriptionallythe expression of IL-2 and other immunoregulatory lymphokines. TheLFA-3/CD2 interaction induces tyrosine phosphorylation of severalintracellular second messengers, Ca²⁺ mobilization, and cAMP production,resulting in elaboration of a variety of cytokines, notably IL-2 andIFN-γ (6). Thus, it appears that the three costimulatory molecules couldbe cooperating by enhancing the antigen-dependent activation of T cells,as well as their production of and response to autocrine and paracrinegrowth factors.

In conclusion, this invention demonstrates for the first time theability of vectors to introduce three or more costimulatory moleculesinto a cell, and to rapidly and efficiently activate both CD4⁺ and CD8⁺T-cell populations to levels far greater than those achieved when one ortwo of these costimulatory molecules is used. This new threshold ofT-cell activation has broad implications in vaccine design anddevelopment.

The effect of the triad of costimulatory molecules on DCs was completelyunexpected. DCs are known by those skilled in the art as the most potentAPC. The data presented in this invention demonstrates that when DCs areinfected with the “Tricom” vector, their ability to activate T-cellsincreases dramatically. These studies demonstrate for the first timethat a DC is not the most potent APC.

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1.-74. (canceled)
 75. A method of enhancing an immune response in anindividual comprising administration of a recombinant vector in anamount sufficient to enhance the immune response, wherein therecombinant vector comprises at least one nucleic acid sequence encodingB7, ICAM-1, and LFA-3.
 76. The method according to claim 75 wherein aroute of administration is intravenous, subcutaneous, intralymphatic,intratumoral, intradermal, intramuscular, intraperitoneal, intrarectal,intravaginal, intranasal, oral, via bladder instillation, or viascarification.
 77. The method according to claim 75 wherein the enhancedimmune response is a cell mediated or humoral response.
 78. The methodaccording to claim 75, wherein the enhancement is of CD4+ T cellproliferation, CD8+ T cell proliferation, or combination thereof. 79.The method according to claim 75, wherein the enhancement is of CD4+ Tcell function, CD8+ T cell function or combination thereof.
 80. Themethod according to claim 75, wherein the enhancement is in IL-2production, IFN-γ production or combination thereof.
 81. The methodaccording to claim 75, wherein the enhancement is of antigen presentingcell proliferation, function or combination thereof. 82.-106. (canceled)107. The method according to claim 75, wherein the recombinant vectorfurther comprises a multiplicity of promoters.
 108. The method accordingto claim 75, wherein the promoters are derived from a eukaryotic source,prokaryotic source, or viral source.
 109. The method according to claim75, wherein the promoters are selected from the group consisting of anSV40 early promoter, RSV promoter, adenovirus major late promoter, humanCMV immediate early I promoter, poxvirus promoter, 30K promoter, I3promoter, sE/L promoter, 7.5K promoter, 40K promoter, and C1 promoter.110. The method according to claim 75, wherein the recombinant vector isselected from the group consisting of poxvirus, adenovirus, Herpesvirus, alphavirus, retrovirus, picornavirus, and iridovirus.
 111. Themethod according to claim 75, wherein the recombinant vector is arecombinant poxvirus.
 112. The method according to claim 111, whereinthe recombinant poxvirus is orthopox, avipox, capripox, or suipox. 113.The method according to claim 112, wherein the recombinant poxvirus isavipox and is selected from the group consisting of fowlpox, canary pox,and derivatives thereof.
 114. The method according to claim 112, whereinthe recombinant poxvirus is orthopox and is selected from the groupconsisting of vaccinia, vaccinia-Copenhagen strain, vaccinia-Wyethstrain, NYVAC, vaccinia-MVA strain, raccoon pox, and rabbit pox. 115.The method according to claim 75, wherein the recombinant vector furthercomprises a foreign nucleic acid sequence encoding at least one targetantigen or immunological epitope thereof.
 116. The method according toclaim 115, wherein the foreign nucleic acid sequence encodes at leastone target antigen comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2 through SEQ ID NO:
 40. 117. The methodaccording to claim 115, wherein the foreign nucleic acid sequenceencodes at least one target antigen selected from the group consistingof a tumor specific antigen, tumor associated antigen, tissue-specificantigen, bacterial antigen, viral antigen, yeast antigen, fungalantigen, protozoan antigen, and parasite antigen, and mitogen.
 118. Themethod according to claim 117, wherein the target antigen is a bacterialantigen derived from a bacterium selected from the group consisting ofChlamydia, Mycobacteria. Legionella, Meningiococcus, Group AStreptococcus, Hemophilus influenzae, Salmonella, and Listeria.
 119. Themethod according to claim 117, wherein the target antigen is a viralantigen derived from a virus selected from the group consisting ofLentivirus, Herpes virus, Hepatitis virus, Orthomyxovirus, andPapillomavirus.
 120. The method according to claim 119, wherein viralantigen is derived from Lentivirus and the Lentivirus is HIV-1 or HIV-2.121. The method according to claim 119, wherein the viral antigen isderived from Herpes virus and the Herpes virus is HSV or CMV.
 122. Themethod according to claim 119, wherein the viral antigen is derived fromHepatitis virus and the Hepatitis virus is Hepatitis A, Hepatitis B,Hepatitis C, Hepatitis D, or Hepatitis E.
 123. The method according toclaim 119, wherein viral antigen is derived from orthomyxovirus and theorthomyxovirus is influenza virus.
 124. The method according to claim117, wherein the target antigen is a tumor associated antigen, tumorspecific antigen, or tissue-specific antigen selected from the groupconsisting of CEA, MART-1, MAGE-1, MAGE-3, GP-100, MUC-1, MUC-2, pointedmutated ras oncogene, normal or point mutated p53, overexpressed p53,CA-125, PSA, C erb/B2, BRCA I, BRCA 11, PSMA, tyrosinase, TRP-1, TRP-2,NY ESO-1, TAG72, KSA, HER-2/neu, bcr-abl, pax3-fkhr, ews-fli-1, modifiedTAAs, splice variants of TAAs, functional epitopes, and epitope agoniststhereof.
 125. The method according to claim 124, wherein the antigen isCEA comprising CEA (6D) having aspartic acid at amino acid position 576.126. The method according to claim 124, wherein the antigen is PSA orPSMA.
 127. The method according to claim 124, wherein the antigen isMUC-1 encoded by a truncated MUC-1 gene consisting of a signal sequence,ten copies of a tandem repeat sequence, and a 3′ coding sequence. 128.The method according to claim 117, wherein the target antigen is a yeastor fungal antigen derived from a yeast or fungus selected from the groupconsisting of Aspergillus, Nocardia, Histoplasmosis, Candida, andCryptosporidia.
 129. The method according to claim 117, wherein thetarget antigen is a parasitic antigen derived from a Plasmodium species,Toxoplasma gondii, Pneumocystis carinii, Trypasosoma species, orLeishmania species.
 130. The method according to claim 75, wherein therecombinant vector further comprises a selectable marker.
 131. Themethod according to claim 130, wherein the selectable marker is selectedfrom the group consisting of lacZ gene, thymidine kinase, gpt, GUS and avaccinia K1L host range gene.